Bio-specific and bio-orthogonal chemistries to switch-off the quencher of a FRET-based fluorescent probe: Application to living-cell biothiol imaging

Article abstract of DOI:10.1039/c4cc03548h

We report the first molecular system that is responsive to both a bio-specific and a bio-orthogonal stimulus. This dual activation process was applied to the design of a biothiol-specific FRET-based fluorescent probe that could be turned-on via an original concept of quencher bleaching. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc03548h

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Bio-specific and bio-orthogonal chemistries to
switch-off the quencher of a FRET-based
fluorescent probe: application to living-cell
biothiol imaging†
Cite this: Chem. Commun., 2014,
0, 10049
5
Received 11th May 2014,
Accepted 3rd July 2014
a
a
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a
C. Egloff, S. A. Jacques, M. Nothisen, D. Weltin, C. Calligaro, M. Mosser,
DOI: 10.1039/c4cc03548h
b
a
J.-S. Remy and A. Wagner*
www.rsc.org/chemcomm
We report the first molecular system that is responsive to both a should demonstrate water solubility, water stability, fast reaction
bio-specific and a bio-orthogonal stimulus. This dual activation kinetics at low concentration, bio-specificity and low toxicity. These
process was applied to the design of a biothiol-specific FRET- stringent operating constraints as well as the precise bio-selectivity
based fluorescent probe that could be turned-on via an original window required for such dual mode of activation make their
concept of quencher bleaching.
development an attractive challenge for organic chemists.
In this communication, we report our study on N-(dicyano-
The field of chemistry in living organisms has long been methylene)aniline oxide that we found to be bio-specifically
focused on fine tuning of structural motifs’ reactivity in order reactive towards endogenous biothiols and bio-orthogonally
to achieve chemo-selective reaction with biological substrates reactive towards sodium dithionite, a bio-compatible exogenous
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such as an endogenous reactive metabolite, an amino acid, a DNA reagent. We also found that reaction with any of the above
sequence, a ROS or acidic pH. This type of biospecific chemistry stimuli resulted in a strong hypsochromic shift of N-(dicyano-
has led to important developments in various domains such as methylene)aniline absorption. Based on this combination of
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imaging probes, pathology specific pro-drugs or advanced bio- chemo and bio-functionalities, we report a novel concept of a
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materials. The idea of using exogenous reactants to trigger a FRET probe which can be activated by either bio-specific or bio-
chemical reaction in a living organism (bio-orthogonal reaction) orthogonal bleaching of its quencher (Fig. 1).
appeared more recently and has nevertheless gained much
Pro-fluorescent probes in complex biological media are acti-
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interest. Among the landmark studies, Meggers and later vated via bio-specific reactions involving biothiols such as Michael
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Bradley showed that organometallic catalysts were able to mediate additions, aromatic nucleophilic additions, thiazolidine ring
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intracellular bond breaking and bond forming processes. Likewise, formation, sulfonamide or sulfonate ester cleavage, disulfide
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Bertozzi, Weissleder, and others pioneered the in vivo use of bio- cleavage and addition–elimination that activates FRET-based
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orthogonal Cu-free click reactions.
probes.
It is noteworthy that even though the reported activation
An attractive goal to extend the scope of such bio-functional
chemistries would now be to combine into a single molecular mechanisms have allowed some degree of biospecific activation
system both the capabilities to react with an endogenous meta-
bolite (bio-specific reaction) and an exogenous reagent (bio-
orthogonal reaction). Such an endeavour relies on the chemist’s
ability to tweak the reactivity of functional groups and reagents in
order to make them functioning under the particular constraints of
complex biological media. For instance, such a molecular system
a
Laboratory of Functional Chemo Systems, UMR 7199, CNRS-UdS, LABEX
MEDALIS, UMR 7199 UdS/CNRS, Faculty of Pharmacy, 74 Route du Rhin,
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7401 Illkirch-Graffenstaden, France. E-mail: alwag@unistra.fr;
Fax: +33-368-854-297
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Laboratory V-SAT, UMR 7199, CNRS-UdS, LABEX MEDALIS, UMR 7199 UdS/CNRS,
Fig. 1 Principle of a FRET-based probe with a biologically and chemically
bleachable quencher. Metabolite-specific reaction shifts the quencher’s
absorption towards lower wavelengths and thus switches fluorescence on.
Further treatment with a bio-compatible exogenous reagent results in complete
bleaching of the quencher and consequently reveals the inactivate probes.
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4 Route du Rhin, 67401 Illkirch-Graffenstaden, France
Phytodia SAS, P oˆ le API, Boulevard Sebastian Brant, 67412 Illkirch-Graffendstaden,
France
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Electronic supplementary information (ESI) available. See DOI: 10.1039/c4cc03548h
This journal is ©The Royal Society of Chemistry 2014
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Fig. 2 Structure of nitrone 1 and absorption spectra of 1 (50 mM) upon
addition of Cys (1 mM) in phosphate buffer (0.1 M, pH 7.4) at 1, 5, 10, 30, 50,
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0 and 100 min.
Fig. 3 Synthetic pathway for the formation of probe 7. Reagents and con-
2 3
ditions: (a) NaNO , HCl, CH OH, 0 1C, 30 min - 30 1C, 4 h (38%); (b) NaOH,
to be obtained, none of them is compatible with subsequent
activation with an exogenous bio-orthogonal reagent. Following HOBT, Et
CH OH, RT, overnight (87%); (c) TCN, DMF, RT, overnight (15%); (d) EDC,
3
3
N, DMF, RT, overnight (27%).
reactivity screening, we have found that N-(dicyanomethylene)-
aniline oxide 1 (see Fig. 2), synthesized from nitroso aniline and
tetracyanoethylene, exhibits suitable features to become an
concentration-dependent increase in fluorescence response was
recorded. We calculated the detection limit for thiols of 50 nM
1
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effective chemo-bio-bleachable quencher.
Firstly, nitrone 1 showed a maximum absorption at lmax
52 nm (Fig. S1, ESI†). Such a high maximum absorption
(Fig. S8, ESI†). We also measured a quantum yield of fluorescence
=
of 15% upon activation with cysteine (Fig. S12, ESI†). Bio-specific
and bio-orthogonal activation of probe 7 was then studied in living
cells. Probe 7 (40 mM) was incubated with normal embryonic fibro-
blast 3T3 cells for 1 h and with 3T3 cells, pre-treated with a-lipoic
acid (500 mM) for 48 h. The purpose of a-lipoic acid (ALA) is to
5
wavelength overlaps with the emission spectrum of the tetra-
methylrhodamine (TAMRA), a widely used fluorophore that
absorbs at lmax = 550 nm and emits at lmax = 580 nm. Secondly,
we found that upon treatment of nitrone 1 (50 mM) with Cys
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increase the intracellular level of reduced GSH. Fluorescence
(1 mM) in phosphate buffer (0.1 M, pH 7.4) the absorption band
emission was measured using a microplate reader (Fig. 4).
A sevenfold increase of intracellular fluorescence intensity was
observed in ALA treated cells compared to non-treated cells.
Then, ALA-3T3 cells were further treated with N-methylmaleimide
at 552 nm almost instantaneously disappeared with the con-
comitant and transient appearance of a new absorption band at
442 nm corresponding to the formation of a Cys-adduct (Fig. 2).
This shift of the maximum absorption peak would represent a
good fluorescence turn-on process of a nitrone-TAMRA FRET
probe. Along with this change in absorption, a solution of
nitrone 1 turned from dark purple to colourless (Fig. S4, ESI†).
Thirdly, the absorbance of compound 1 was instantaneously
neutralized upon treatment with sodium dithionite in phosphate
buffer (0.1 M, pH 7.4). This would be a smooth chemo-bleaching
process of the corresponding nitrone-TAMRA FRET probe. Finally,
MTT assays showed no inherent toxicity of nitrone 1 in HaCaT
cells under physiological conditions (Fig. S5, ESI†).
Synthesis of the nitrone-TAMRA FRET probe 7 started with the
preparation of intermediate 2 (Fig. 3). Amine alkylation between
N-methylaniline and methyl-4-bromobutyrate gave compound 2.
Nitrosation with sodium nitrite led to nitroso derivative 3. After
saponification, 4 reacted with tetracyanoethylene (TCN) to give 5.
Finally, coupling of 5 with TAMRA amine 6 gave the turn-on
fluorescent probe 7 after purification by semi-preparative HPLC.
To assess the chemoselectivity of N-(dicyanomethylene)-
aniline oxide moiety activation, probe 7 (1 mM) was treated
with various biological analytes (5 mM) including amino acids,
oxidizing and reducing agents, metal ions, glucose, nucleosides,
ascorbic acid and thiols (Cys, Hcy, and GSH) in phosphate buffer
(
NMM) to decrease the level of free thiols within the cells by
electrophilic alkylation and with buthionine sulfoximine (BSO),
a specific inhibitor of g-glutamylcysteine synthetase and therefore
a GSH synthesis inhibitor. Under both conditions fluorescence
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intensity was found to be equivalent to that of untreated cells,
thus indicating that activation of probe 7 was indeed descriptive
of the intracellular level of free biothiols.
To validate the second mode of activation of probe 7, namely
bio-orthogonal chemical bleaching, sodium dithionite (10 mM)
was added to each one of the above-described assays (Fig. 5).
Finally, cells treated with probe 7 were imaged using con-
focal laser scanning microscopy. Cells were treated with blue
(0.1 M, pH 7.4) (Fig. S10, ESI†). The results demonstrated a
Fig. 4 Fluorescence of probe 7 (1 mM) incubated for 1 h with 3T3 cells,
significant fluorescence increase at 580 nm only upon reaction treated under various conditions: CTL = untreated 3T3 cells; 7 = probe 7
with non-treated cells; 7 + ALA = probe 7 with cells pre-treated for 48 h
with ALA (500 mM); 7 + ALA + NMM = probe 7 with cells pre-treated for
with thiol derivatives. With all the other analytes, no real fluores-
cence emission enhancement was observed. This thiol-specific
reactivity was further confirmed by inhibiting the reaction with
iodoacetate, a thiol-alkylating agent (Fig. S11, ESI†). Next, our
4
7
8 h with ALA (500 mM), then treated for 30 min with NMM (500 mM);
+ ALA + BSO = probe 7 with cells pre-treated for 48 h with ALA (500 mM)
then treated for 30 min with BSO (100 mM). Data were measured using a
probe 7 was reacted with increasing equivalents of cysteine and a microplate reader (excitation filter 530/25 nm; emission filter 590/35 nm).
10050 | Chem. Commun., 2014, 50, 10049--10051
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combination of bio-functional reaction and quencher-based
fluorescence activation expands the repertoire of the turn-on/off
probe mechanism. It also illustrates one of the many-to-come
possibilities of using biofunctional chemistry to investigate bio-
logical systems with compact and multi-functional molecular tools.
We are grateful for the financial support from R ´e gion Alsace
and International Centre for Frontier Research in Chemistry
(ICFRC). We thank Dr A. Klymchenko and Dr A. Specht for
helpful discussion.
Notes and references
Fig. 5 Fluorescence of 7 in 3T3 cells under various conditions before
(green) and after (red) addition of dithionite (10 mM). CTL = untreated 3T3
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1
In summary, we have reported the first molecular system
that is responsive to both a bio-specific and a bio-orthogonal
stimulus. This was visualized via activation of a FRET probe
through an original mechanism of quencher bleaching. Such a 16 O. W. Griffith and A. Meister, J. Biol. Chem., 1979, 254, 7558.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 10049--10051 | 10051