Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes

Article abstract of DOI:10.1039/d0cc01512a

A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes¹O

Full text of DOI:10.1039/d0cc01512a

ChemComm
View Article Online
View Journal
COMMUNICATION
Microscope laser assisted photooxidative
activation of bioorthogonal ClickOx probes†
Cite this:DOI: 10.1039/d0cc01512a
Attila Kormos, ‡ Dora Kern,‡ Alexandra Egyed, Bianka Soveges,
´
¨
Received 26th February 2020,
Accepted 6th April 2020
Krisztina Nemeth
and Peter Kele
*
´
´
DOI: 10.1039/d0cc01512a
rsc.li/chemcomm
A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe a bioorthogonal function, via genetic code expansion, enables
was synthesized and studied in light-assisted, bioorthogonal labeling much greater flexibility in terms of labeling sites and highly
schemes. Experimental results revealed that the bioorthogonally con- selective installation of small, minimally perturbing synthetic
jugated probe efficiently sensitizes 1O2 generation upon illumination probes carrying a complementary bioorthogonal motif.4 A further
with green or orange light and undergoes self-oxidation leading to an advantage of bioorthogonal labeling schemes is that they are not
intensely fluorescent sulfoxide product. An added value of the present limited to biomolecules directly encoded in the genome and they
probe is that it is also suitable for STED super-resolution microscopy allow probing of nucleic acids, lipids, sugars, etc. as well. One of
using a 660 nm depletion laser.
the most popular bioorthogonal reactions in labeling schemes is
the inverse electron demand Diels–Alder reaction of tetrazine
Due to recent developments, fluorescence microscopy has become dienes and strained dienophiles (e.g. cyclooctynes and trans-
the primary technique for the visualization of cellular structures cyclooctenes).5 An extra feature of the tetrazine moiety, besides
and events because of its excellent sensitivity and spatiotemporal being an excellent bioorthogonal platform, is its capability of
resolution. Thus, it has grown into an inevitable tool in studies quenching fluorescence of fluorophores. This feature can be
aiming at understanding biological processes.1 Its importance has harvested in the development of turn-on (fluorogenic) probes as
become even more profound since the advent of super-resolution a chemical transformation of the tetrazine motif results in
techniques. The emerging subdiffraction methods enable reinstated emission intensity (reactivity-based fluorogenicity).6
visualization of biological structures in details never seen before.2 When it comes to the design of such bioorthogonally applicable
Parallel to the evolution of optical microscopy techniques, chemical small synthetic probes, features such as suitable excitation and
biology methods have also gone through remarkable developments, emission maxima, brightness, quantum yields, photostability,
which led to advanced labeling techniques.3 Fusion of engineered membrane permeability, etc. are all on the wish list.7a Besides
fluorescent proteins (FPs) to the protein of interest (POI) is routinely these, improved probes should also address problems often
accomplished and enables highly selective labeling; however, the associated with fluorescence imaging using small, synthetic
photophysical characteristics of FPs often limit resolution. Though probes. Such problems are auto- and background fluorescence.7
the use of small, chemically tailored probes with improved photo- While the former one is relatively easily addressed by selecting
physical features is also possible through fusion tags (e.g. halo-, frames excitable towards the red end of the spectrum, or having
snap- or clip-tags), similar to FPs, the comparable size of these large Stokes shifts, reduction of background fluorescence either
tags often hampers the native function of the POI. Furthermore, requires extensive washing cycles or, more elegantly, the use of
they also impair precise localization of the protein of interest fluorogenic probes, whose fluorescence intensity increases
due to linkage error. Moreover, the use of FPs and other fusion remarkably upon selective ligation to the biomolecule of interest.
tags is limited (mostly) to the terminal labeling of proteins only. We and others recently reported on improved probes, where the
Site-specific incorporation of non-canonical amino acids bearing two-in-one feature of the tetrazine motif, i.e. being bioorthogonal
and a quencher of fluorescence at the same time, was exploited in
Chemical Biology Research Group, Institute of Organic Chemistry, Research Centre
the development of bioorthogonally applicable fluorogenic
probes.6b,8 It was observed, however, that the fluorescence
increase upon bioorthogonal ligation, i.e. fluorogenicity, dramatically
decreases towards the biologically preferred red range of the spec-
trum. In order to access better fluorogenic probes in this spectral
range, we have introduced the concept of multiple fluorogenicity,
´
for Natural Sciences, Magyar tudosok krt. 2, Budapest H-1117, Hungary.
E-mail: kele.peter@ttk.hu
† Electronic supplementary information (ESI) available: Details of synthetic proce-
dures and characterization data, spectroscopy experiments and protocols of actin-
labeling and microscopy imaging experiments. See DOI: 10.1039/d0cc01512a
‡ These authors contributed equally to this work.
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Communication
ChemComm
Table 1 Main spectral characteristics of probes 1 and 10 and their click-
where we either increased the number of quencher units (e.g. two
tetrazines)9 or combined different quenching mechanisms10 (e.g.
tetrazine and polarity driven valence tautomerism). Though these
multiply fluorogenic probes have improved fluorogenicities in
the red regime, their applicability is limited either to special
conjugatesa
lexc
lem
FBCN
Ftet
/
(nm) (nm) e (MÀ1 cmÀ1
)
F
F10.BCN/F1
1a
608
608
554
743
743
588
586
743
743
584
583
31 600
32 100
41 200
42 900
39 300
37 100
35 600
35 800
0.0037 1.19
0.0044
154
sequences or microenvironments. Thus, new approaches that 1a.BCN
10a
0.31
0.57
1.84
could facilitate the design of generally applicable, visible-light
excitable probes with improved fluorogenicity are still needed. In
10a.BCN 553
1b
610
607
554
0.0036 1.17
0.0042
158
the quest of improved fluorogenic probes, we searched for 1b.BCN
10b
0.21
0.57
2.7
alternative mechanisms that can strengthen the moderate reactivity
10b.BCN 551
based fluorogenicity of tetrazinylated probes in the green-red range.
During tetrazine-based ligation schemes, the highly electron
deficient tetrazine function is transformed into a dihydropyrida-
zine or pyridazine, depending on the dienophile. Such fine changes
a
Complete consumption of tetrazines and formation of BCN conjugates
were monitored by LC-MS.
in electronic properties can promote profound differences in reactivity-based fluorogenicity is very poor in this spectral range
reactivities, e.g. in oxidizability, between reacted and non- (Table 1). However, a considerable blue-shift of spectral maxima and
reacted forms. More particularly, we intended to use light to a two-orders of magnitude increase in emission intensities were seen
induce such oxidation. We further envisioned that transformation of in the case of the oxidized forms compared to their respective
an appending tetrazine moiety to a pyridazine, upon bioorthogonal reduced congeners, irrespective of being clicked or not. Next, we
ligation, leads to a less electron deficient congener that is more elaborated whether the oxidized species, 10, and their BCN con-
susceptible to light-induced oxidation. This holds promise for the jugates can be accessed upon irradiation of probes 1 with light. To
primary switchability of the specifically reacted, i.e. biomolecule- this end, we have illuminated oxygenated solutions (methanol
conjugated, forms, enabling facile distinction from non-specifically was used as solvent to obtain a reasonable concentration range
bound, unreacted species, which overall translates to a new fluoro- necessary for the photophysical studies) of 1a and b and their
genic mechanism. Prompted by this, we sought frames whose respective in situ formed BCN conjugates with orange (around
structure is likely to be oxidized upon encountering light resulting 600 nm) and green (around 540 nm) LEDs (for the LED emission
in altered photophysical properties. Phenothiazines, e.g. chlor- spectra, see Fig. S1, ESI†). To our delight, in both cases, the
promazine or methylene blue, are known for their singlet oxygen formation of the respective oxidized products was evident, as
generating properties upon irradiation with light.11 Oliveros et al. indicated by LC-MS and the emission spectra (Fig. S2, ESI†). In
reported on the photooxidation of 10-methyl phenothiazine. Their the presence of a known 1O2 scavenger, sodium azide, however,
study revealed that the process involved self-sensitized 1O2 leading no photooxidation was observed (Fig. S3, ESI†).14 At the same
to 10-methyl phenothiazine sulfoxide, possessing distinct spectral time, no oxidation occurred when deoxygenated samples were
maxima.12a Other instances reported on sensitizer (e.g. Rose- illuminated or when oxygenated samples were kept in the dark.
Bengal)-mediated photoxidation of N-ethylphenothiazine.12b To These results confirm that light-triggered production of 1O2
test our hypothesis, we designed rhodamine fused phenothiazines sensitized by probes 1a and 1b or their BCN conjugates is
1a and b (Fig. 1, and ESI†).13 We have also prepared sulfoxides 10a responsible for the oxidation process (Fig. S4, ESI†). The formation
and 10b for control experiments. With the probes in hand, we next of 1O2 was further confirmed using the RNO method (Fig. S5,
examined their spectral properties together with their in situ ESI†).15 We compared the photooxidative characteristics of probes
formed click-reaction products with a strained cyclooctyne 1a and 1b with those of their BCN-conjugates. The gratifying results
((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol, BCN). Not much showed a marked difference between the photoconversion rate of
difference in intensities could be seen between the click-conjugates the clicked conjugates and the parent tetrazines. For example, when
and their parent tetrazines, indicating that tetrazine-dependent, probe 1a was irradiated with green light for 2 h, only a 20%
photoproduct could be observed. However, when click conjugate
1a.BCN was irradiated with the same light source for 2 h, 90% of the
starting material was converted to the respective oxidized product,
as indicated by LC-MS (Fig. S6, ESI†). This confirms our hypothesis
regarding electronic changes upon click reaction. Upon prolonged
irradiation of 1, the appearance of so called dealkylated photobluing
products16 together with unidentifiable byproducts was observed
(Fig. S7 and Table S1, ESI†).
The finding that BCN conjugated products were more prone
to photooxidation, leading to a product emitting around 600 nm
upon LED illumination, can be translated to selective photo-
oxidation of specifically click-conjugated probes, 1a and b, in
cellular labeling schemes. This implies a special fluorogenic
Fig. 1 Structures of probes 1 and 10.
mechanism as unreacted or non-specifically bound probes
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

View Article Online
ChemComm
Communication
remain mainly in their weakly emissive reduced form, emitting
above 650 nm, thus minimally contributing to background fluores-
cence upon microscope imaging in the 565–635 nm range, even
under no-wash conditions. Upon examination of the excitation and
emission bands of 1 and 10.BCN (Fig. 2 and Fig. S8, ESI†), we
envisioned that the commercially available green laser (552 nm)
used in confocal microscopes is suitable to promote photooxidation
and subsequent excitation of the photoproducts for imaging.
Excitation in the orange range is also suitable to carry out the
oxidation step, however the product requires green excitation.
Thus, excitation with a green laser seems an ideal two-in-one
combination.
These considerations prompted us to carry out a proof-of-
principle study with probe 1. Towards this end, bundles of actin
filaments of fixed COS7 cells were tagged with a cyclooctynylated
(BCN) affinity tag, BCN-phalloidin. Excess of the affinity tag was
removed, then the samples were stained with probe 1a and
washed. Cells were then imaged under a confocal microscope.
Excitation at 638 nm with a 652–760 nm detection window
(i.e. the spectral range suitable to excite and detect probe 1a)
resulted in poor image quality with no structural details. Similar
image quality was obtained when the same detection window was
set at 552 nm excitation. However, when the samples were excited
at 552 nm and detected between 565 and 653 nm (i.e. the spectral
range suitable to image probe 10), nice images of the actin
filament network appeared immediately. These results confirm that
the green laser used for excitation is suitable both for immediate
photooxidation and subsequent excitation of the specifically con-
jugated, oxidized probes. Next, we wished to explore whether the
marked difference in photooxidizability between the reacted and
unreacted forms seen in solution allows selective activation of the
Fig. 3 Actin filament staining. COS7 cells were tagged with cyclooctynylated
phalloidin, then labeled with 1a (scale bar: 10 mm).
Fig. 4 Confocal and STED images of BCN-phalloidin tagged actin fila-
ments stained with 1a (lexc = 552 nm, and lSTED = 660 nm (CW); FWHM:
374 Æ 127 vs. 139 Æ 9 nm for confocal and STED images, respectively).
specifically conjugated probes. Therefore, we repeated the above images to the washed samples. This indicates that primarily,
experiment; however, at this time, excess of probe 1a was not the clicked probes are oxidized by the laser. We also carried out
washed out (Fig. 3). The gratifying results showed similar the labeling experiments with the oxidized form, 10a. As
expected, low quality images of the actin network were acquired
due to the considerable background fluorescence of the non-
specifically bound, unreacted probes (Fig. S11, ESI†). These
observations further confirm that the specifically bound, clicked
products are selectively photooxidized to result in intensely
fluorescent products by the green laser, while unreacted tetra-
zine probes mostly remain in their poorly fluorescent reduced
form contributing minimally to background fluorescence. The
emission spectrum of 10a.BCN (Fig. 2) suggests that such
clicked, oxidized probes (ClickOx) could even be suitable for
super-resolution, STED microscopy imaging using a commercial
660 nm depletion laser. Indeed, when we tested the cells stained
with probe 1a at their actin filament network, subdiffraction
resolution was achieved with STED microscopy using a 552 nm
laser for excitation and a CW 660 nm laser for depletion (Fig. 4).
In conclusion, we have designed and synthesized bioorthogonally
applicable, tetrazine functionalized rhodaphenothiazines and
elaborated their light-assisted photooxidation-based fluorogenic
characteristics. Experimental results suggest that the probes
Fig. 2 Excitation and emission spectra of 1a and 10a-BCN in PBS-SDS
with the LED emission ranges used for the photooxidation experiments
efficiently sensitize 1O2 generation upon illumination with
green or orange light. Furthermore, the produced singlet oxygen
promotes self-oxidation of the probes leading to intensely
together with commercial microscope laser lines (note that the spectra of
1a are magnified 20Â for better comparison).
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Communication
ChemComm
2 (a) J. Vangindertael, R. Camacho, W. Sempels, H. Mizuno, P. Dedecker
and K. P. F. Janssen, Methods Appl. Fluoresc., 2018, 6, 022003;
(b) D. Baddeley and J. Bewersdorf, Annu. Rev. Biochem., 2018, 87,
965–989; (c) G. Huszka and M. A. M. Gijs, Microsyst. Nanoeng., 2019, 2,
7–28; (d) S. J. Sahl, S. W. Hell and S. Jakobs, Nat. Rev. Mol. Cell Biol., 2017,
18, 685–701.
3 (a) C. Li, A. G. Tebo and A. Gautier, Int. J. Mol. Sci., 2017, 18, 1473;
(b) G. Zhang, S. Zheng, H. Liu and P. R. Chen, Chem. Soc. Rev., 2015,
44, 3405–3417.
fluorescent sulfoxide products. Further studies revealed a marked
difference between the photooxidizability of the free tetrazine
and the bioconjugated pyridazine forms, which implies a two-
orders of magnitude fluorescence increase due to this new
fluorogenic mechanism. This unique fluorogenic behavior allows
selective photoactivation of specifically conjugated probes, as
demonstrated in the labeling schemes of actin filaments with
low background fluorescence even under no-wash conditions.
A remarkable feature of the developed ‘‘ClickOx’’ probes is that
the same commercial green excitation laser (552 nm) is suitable
for carrying out photooxidation of the probes and subsequent
excitation of the product. An added value of the present probe is
that it is also suitable for STED super-resolution microscopy
using a 660 nm depletion laser. Since it is the lack of availability
of good probes that is considered among the limitations of
further improvements of resolution, we believe that discovery of
new mechanisms and design elements such as the presented
photo-activation mechanism is an important step towards
improved probes. Currently, we are exploring the potential of
the new ClickOx probe in targeted photodynamic therapy and in
light-assisted indication of oxidative stress-triggered inflamma-
tion in cells and tissues, the results of which are to be published
in due course.
´
4 (a) K. Lang and J. W. Chin, Chem. Rev., 2014, 114, 4764–4806; (b) I. Nikic,
´
T. Plass, O. Schraidt, J. Szymanski, J. A. Briggs, C. Schultz and E. A. Lemke,
Angew. Chem., Int. Ed., 2014, 53, 2245–2249; (c) J. W. Chin, Annu. Rev.
Biochem., 2014, 83, 379–408.
5 (a) E. Kozma, O. Demeter and P. Kele, ChemBioChem, 2017, 18, 486–501;
(b) A.-C. Knall and C. Slugovc, Chem. Soc. Rev., 2013, 42, 5131–5142.
6 (a) E. Kozma and P. Kele, Org. Biomol. Chem., 2019, 17, 215–233;
(b) A. Wieczorek, P. Werther, J. Euchner and R. Wombacher, Chem. Sci.,
2017, 8, 1506–1510; (c) J. C. Carlson, L. G. Meimetis, S. A. Hilderbrand
and R. Weissleder, Angew. Chem., Int. Ed., 2013, 52, 6917–6920.
7 (a) L. Wang, M. S. Frei, A. Salim and K. J. Johnsson, J. Am. Chem.
´
Soc., 2019, 141, 2770–2781; (b) G. B. Cserep, A. Herner and P. Kele,
Methods Appl. Fluoresc., 2015, 3, 042001.
8 (a) L. G. Meimetis, J. C. Carlson, R. J. Giedt, R. H. Kohler and
R. Weissleder, Angew. Chem., Int. Ed., 2014, 53, 7531–7534; (b) Y. Lee,
W. Cho, J. Sung, E. Kim and S. B. Park, J. Am. Chem. Soc., 2018, 140,
´
´
974–983; (c) S. J. Siegl, J. Galeta, R. Dzijak, A. Vazquez, M. Del Rıo-
ˇ
Villanueva, M. Dracnisk´y and M. Vrabel, ChemBioChem, 2019, 20,
886–890; (d) G. Knorr, E. Kozma, A. Herner, E. A. Lemke and P. Kele,
Chem. – Eur. J., 2016, 22, 8972–8979; (e) G. Knorr, E. Kozma,
´
¨ ¨
J. M. Schaart, K. Nemeth, G. Torok and P. Kele, Bioconjugate Chem.,
2018, 29, 1312–1318.
The present work was supported by the ‘‘Lendu¨let’’ Program
of the Hungarian Academy of Sciences (LP2013-55/2013) and the
National Research, Development and Innovation Office (NKFIH-
+
´
9 (a) O. Demeter, A. Kormos, C. Koehler, G. Mezo, K. Nemeth,
E. Kozma, L. Takacs, E. A. Lemke and P. Kele, Bioconjugate Chem.,
2017, 28, 1552–1559; (b) A. Kormos, C. Koehler, E. A. Fodor,
+
Z. R. Rutkai, M. E. Martin, G. Mezo, E. A. Lemke and P. Kele,
´
K-123917). DK is grateful for the support of the UNKP-19-2 New
Chem. – Eur. J., 2018, 24, 8841–8847.
10 E. Kozma, G. Estrada Girona, G. Paci, E. A. Lemke and P. Kele,
Chem. Commun., 2017, 53, 6696–6699.
11 (a) T. Iwaoka and M. Kondo, Bull. Chem. Soc. Jpn., 1974, 47, 980–986;
(b) R. W. Redmond and J. N. Gamlin, Photochem. Photobiol., 1999,
70, 391–475.
National Excellence Program of the Ministry for Innovation and
Technology.
12 (a) T. Manju, N. Manoj, A. M. Braun and E. Oliveros, Photochem.
Photobiol. Sci., 2012, 11, 1744–1755; (b) E. Baciocchi, T. Del Giacco,
O. Lanzalunga, A. Lapi and D. Raponi, J. Org. Chem., 2007, 72, 5912–5915.
13 C. Liu, X. Jiao, Q. Wang, K. Huang, S. He, L. Zhao and X. Zeng, Chem.
Commun., 2017, 53, 10727–10730.
Conflicts of interest
There are no conflicts to declare.
14 A. Gollmer, J. Arnbjerg, F. H. Blaikie, B. W. Pedersen,
T. Breitenbach, K. Daasbjerg, M. Glasius and P. R. Ogilby, Photo-
chem. Photobiol., 2011, 87, 671–679.
Notes and references
1 (a) E. A. Specht, E. Braselmann and A. E. Palmer, Annu. Rev. Physiol.,
´
2017, 79, 93–117; (b) P. O. Bayguinov, D. M. Oakley, C. C. Shih, 15 I. Kraljic and S. El Mohsni, Photochem. Photobiol., 1978, 28, 577–581.
ˇ
D. J. Geanon, M. S. Joens and J. A. J. Fitzpatrick, Curr. Protoc. Cytom., 16 A. N. Butkevich, M. L. Bossi, G. Lukinavicius and S. W. Hell, J. Am.
2018, 85, e39.
Chem. Soc., 2019, 141, 981–989.
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020