An original class of small sized molecules as versatile fluorescent probes for cellular imaging

Article abstract of DOI:10.1039/c9cc03765a

An unusual class, compact in size, of fluorescent probes based on pyridazino-1,3a,6a-triazapentalene scaffolds exhibits promising fluorescent properties (quantum yield values up to 73%, large Stokes shifts, emission wavelengths located in the green-yellow

Full text of DOI:10.1039/c9cc03765a

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DOI: 10.1039/C9CC03765A
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An Original Class of Small Sized Molecules as Versatile Fluorescent
Probes for Cellular Imaging
Doina Sirbu,a Julien Diharce,a Ivana Martinić,b Nicolas Chopin,a Svetlana V. Eliseeva,b Gérald
Received 00th January 20xx,
Accepted 00th January 20xx
Guillaumet,a Stéphane Petoud,b Pascal Bonneta and Franck Suzenet*a
DOI: 10.1039/x0xx00000x
An unusual class, compact in sizes, of fluorescent probes based on molecular tools for novel bioimaging applications.8-12 In this
pyridazino-1,3a,6a-triazapentalene scaffolds exhibits highly context, focusing on very low molecular weight/size
fluorescent properties (quantum yield values up to 73%, large fluorophores is of major interest for minimizing
Stokes shifts, emission wavelengths located in the green-yellow pharmacokinetic alterations of the targeting biomolecules to
range, excellent solubility) with very good photostability suitable which they are linked.13 Small dyes may also find specific
interest in ligase fusion proteins.14 Obviously, those
fluorophores have to rely on (i) optimized spectroscopic
properties, including high quantum yield values, large Stokes
shifts and good photostability,15,16 and (ii) biological media
compatibility i.e. good chemical stability, low cytotoxicity, cell
permeability and solubility, in order to be considered as suitable
probes for cellular imaging.
for optical imaging applications.
Fluorescent organic molecules are essential compounds for the
detection, quantification and understanding of biological
processes applied in chemical biology, biochemistry, biomedical
research and diagnostics.1 The main advantages of fluorescent
organic molecules include high versatility, moderate molecular
size and weight, chemical stability, and ability to exhibit
switchable or activatable spectroscopic properties. This
fluorogenic behavior can be initiated by a chemical or
biochemical process according to four main different reaction
mechanisms: pH sensitivity, complexation/decomplexation,
formation or cleavage of a covalent bond and redox reactions.2-
5
Although numerous fluorophores have already been described
in the literature, the diversity of the molecular frameworks of
those commonly used for cell imaging probes is often limited to
coumarin, xanthene (fluorescein, rhodamine, Texas), BODIPY
and cyanine cores.6,7 These dyes cannot be considered as ideal
probes for optical imaging since none of them combine high
fluorescence with optimal absorption and emission
wavelengths, good chemical and photostability, sufficient water
solubility and small molecular weight/size.
Therefore, the discovery of novel fluorophore-based scaffolds
remains an important challenge to offer new highly versatile
platforms providing alternative and/or complementary
Fig 1 Summary of the state of the art in the field of triazapentalene derivatives.
Photophysical properties of 1a,3a,6a-triazapentalene (a) benzotriazapentalene
(b), and novel nitrogen-containing fused ring TAP derivatives (c).
A few years ago, Namba’s team pioneered the fluorescent
properties of the 1,3a,6a-triazapentalene (TAP) derivatives
(Figure 1a)17 and showed that this highly condensed dipolar
structure offers very promising spectroscopic properties and
suitability for biological applications.18,19 To study the impact of
the TAP substituents on spectroscopic properties, the
a. Institut de Chimie Organique et Analytique - ICOA UMR7311, rue de Chartres,
45100 Orléans, France. E-mail: franck.suzenet@univ-orleans.fr
b. Centre de Biophysique Moléculaire CNRS UPR 4301, rue Charles Sadron, 45071
Orléans Cedex 2, France.
Electronic Supplementary Information (ESI) available: Experimental procedures,
characterization data, 1H and 13C NMR spectra, theoretical calculations,
spectroscopic analysis, cellular optical imaging, cytotoxicity tests. See
DOI: 10.1039/x0xx00000x
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development of specific synthetic pathways was required.20-26 nitrogen atoms is particularly relevant as is illustrated with
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Although some improvements of this highly promising TAP compound 3 for which a blue shift oDf OtIh: e10.1a0b3s9o/rCp9tCioCn037a6n5dA
scaffold were observed, further investigations towards fused emission bands was observed.
ring chemical entities have not been considered so far as
Table 1 Spectroscopic characterization of the tricyclic compounds
in DMSO.
1, 2, 3 and
benzotriazapentalene 1 as a fused ring system were already
reported as non-fluorescent (Figure 1b).17
4
[d]
Dye

abs[nm]
[a]

em[nm]
[nm] [c]
max
[e]
Herein, we report the design, the synthesis, the fluorescent
properties and the cellular imaging compatibility of a novel class
of small nitrogen-containing tricyclic frameworks. We
demonstrate the crucial impact of the additional fused
heteroaromatic ring on the TAP system towards promising
spectroscopic properties for such a low molecular weight
structure with excellent chemical stability.
In the course of a research program on the design of energetic
compounds, we identified pyrazolo-triazolo-pyridine27 and
pyrazolo-triazolo-pyrazine scaffolds28 (Figure 2, compounds 2
and 4, respectively). We postulated that the electron deficient
character of the fused azine or diazine cycle could favor an
intramolecular charge transfer (ICT) and promote the emission
of this tricyclic scaffold (Figure 1c).
[b]
1
2
3
4
370
387
362
418
397
446
440
516
27
59
78
98
12700
18700
1200
<0.001
0.018
0.15
15500
0.15
[a] Apparent maxima of absorption bands of the dyes. [b] Apparent maxima
of emission bands of the dye. [c] Stokes Shift values calculated as the
difference between the maxima of absorption and emission bands. [d] Units:
L.mol-1.cm-1. [e]
153 (
=0.38 in EtOH) as a reference standard.31
 is the relative fluorescence quantum yield using Coumarin

In addition, scaffold 4 showed a much higher molar extinction
coefficient compared to compound 3, giving rise to an improved
brightness. The pyrazino-[1a,3a,6a]-triazapentalene 4 stands
out as our model scaffold which could be easily optimized by a
simple and large scope of chemical modulation.
In order to study the effect induced by the presence of nitrogen
atoms in the (di)azine rings on the fluorescent properties, we
managed to synthesize and characterize the tricyclic scaffolds 1,
2, 3 and 4 (Figure 2).
Interestingly, the fluorescent tricyclic scaffold 4 displayed a
good solubility in water (>1 mg.mL-1) along with an apparent
position of the emission maximum larger than 530 nm, a Stokes
shift of approximately 130 nm and a molar extinction coefficient
of higher than 12000 M-1.cm-1. The excellent chemical stability
of dye 4 was also observed from pH4 to pH11 in water solutions.
The solvatochromic behavior of 4 was obtained by recording
absorption and emission spectra in various solvents. Whereas
almost no change in the positions of the apparent absorption
maxima was observed, a strong bathochromic effect was
noticed from apolar to protic polar solvents characterized by a
larger Stokes shift in water (128 nm) compared to chloroform
(85 nm) (see SI for details).
In order to provide gain insights into the effect of nitrogen
additions on the (het)aryl part of the tricyclic molecules (1 to 4),
TD-DFT calculations were carried out10,32 (see SI for details) and
analysis of frontier orbitals for each compound was performed.
The electronic density of the HOMO appeared, in general, to be
weakly impacted by the substitution of a carbon atom by a
nitrogen. The presence of nitrogen on the fused ring decreased
the HOMO electronic density on adjacent atoms without any
impact on the 1,3a,6a-triazapentalene moiety. In contrast, the
pyridine ring in 2 displaced the LUMO electronic density from
the 1,3a,6a-triazapentalene moiety to the 6-member ring,
increasing the positive partial charges at the carbons and the π-
deficient character of the fused ring (see Figure 3 and Table S1).
These two complementary effects may explain the
enhancement of the intramolecular charge transfer (ICT).
Fig. 2 Chemical structures of compounds 1, 2, 3 and 4.
Synthesis of compounds 1,29 227 and 428 was undertaken
following reported protocols and they were isolated in correct
yields (see Supporting Information). Regarding compound 3, an
original synthetic pathway was developed in four steps
involving a key copper catalyzed C-N bond formation30 (see SI
for details). Having prepared the triazapentalene scaffolds 1-4,
we then carried out the complete spectroscopic
characterization (Table 1). The absence of fluorescence for
compound 1 was confirmed.17 The azine scaffold (compound 2)
showed very weak fluorescence in contrast to its diazine
isomers (compounds
3 and 4) which exhibited intense
fluorescence bands with a very promising quantum yield values
of 15% in DMSO.
Furthermore, the spectroscopic data clearly showed that the
number and position of nitrogen atoms in the additional
aromatic ring greatly affects the absorption and emission bands
positions in agreement with the calculations (SI, Table S1).
Interestingly, the presence of an extra nitrogen atom in
compound 2 induced 17 nm and 49 nm red shifts for absorption
and emission bands respectively compared to compound 1. This
trend is confirmed with a pyrazine-containing fused aromatic
ring (compound 4) with a remarkable bathochromic effect for
both absorption and emission bands offering a very compact
organic fluorescent scaffold with an emission maximum at 516
nm. It should be noted that the position of these two additional
Figure 3. Major atomic contribution to the LUMO density for
compounds 1 to 4.
2 | J. Name., 2012, 00, 1-3
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The pyrazine ring in 4 slightly emphasized the effect on the triazapentalenes were obtained through a Suzuki cross-coupling
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LUMO atomic contribution compared to 1 while the pyrimidine reaction following pathway B (Scheme 1,DsOeIe: 1S0I.1f0o3r9d/Ce9tCaiCls0)3.765A
ring in 3 did not induce the same behavior. Indeed, by
Table 2. Spectroscopic characterization of tricyclic analogs of (pyrazinyl)-1a,3a,6a-
comparing 3 and 4, the LUMO density is less localized on the
triazapentalene and –tetrazapentalene in CH2Cl2 and DMSO.
pyrimidine part than on the pyrazine. Consequently, the

abs[nm]
[a]

em[nm]
[b]
enhanced π-electron deficient character of the pyrazine could
Dye
Solvent
max[c]
[d]
Scheme 1 Synthesis of various diversified pyrazinyl-1a,3a,6a-triazapentalenes
4
CH2Cl2
CH2Cl2
415
494
13000
12700
0.42
0.51
from chloropyrazine
5 (pathway A) and from organometallic coupling processes
with halogeno-triazapentalene 4i or 4j (pathway B).
4a
391
427
4b
4c
CH2Cl2
DMSO
CH2Cl2
DMSO
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
410
436
410
415
416
409
403
418
414
412
418
417
417
406
419
418
418
470
529
512
521
473
467
464
473
483
487
496
497
491
518
485
484
477
12133
13200
7105
0.37
0.03
0.08
0.03
0.023
0.52
0.034
0.73
0.27
0.27
0.45
0.29
0.38
0.04
0.48
0.52
0.51
4d
13700
24157
14911
14706
11470
16443
19300
13781
18185
15085
16151
14200
14711
11500
4e
4f
4g
4h
4i
4j
6a
6b
6c
6d
6e
6f
6g
[a] Apparent maxima of absorption bands. [b] Apparent maxima of emission
band. [c] Units: L.mol-1.cm-1. [d]
is the relative fluorescence quantum yield
estimated by using Coumarin 153 (
=0.38 in EtOH) as a reference standard.31


As shown in Table 2, the presence of an extra nitrogen on the
azole ring (for compounds 4a and 4b) produced a strong
hypochromic effect without affecting the other spectroscopic
properties (ϕ and ). In contrast, an electron donating group
wherever the position (compounds 4c, 4d and 6d) produced a
bathochromic effect, while being detrimental for the quantum
yield. However, an electron-withdrawing group (CF3, CO2Et,
ArCF3 and ArCO2Et for compounds 4f, 4h, 6f and 6e respectively)
was highly beneficial to the quantum yield (up to 73% in CH2Cl2
for compound 4h) when attached in position 3 of the azole. The
molar extinction coefficient was greatly improved when the
ester group was introduced in position 2. Finally, it is
noteworthy that excitation and emission wavelengths were
almost unaffected by the presence of an aryl group on the azole
moiety (6a vs 4).
In order to demonstrate the ability of the novel triazapentalene
compounds to be used as fluorescence probes for the imaging
of living cells, epifluorescence microscopy experiments were
performed on HeLa cells incubated with dye 6e. The resulting
images suggest that this dye can be taken up by cells and the
signal in the green region of the visible spectrum was recorded
upon excitation using a 414 nm band pass 46 nm filter (see SI,
Figure S9, Top) whereas, a minimal autofluorescence signal was
observed for untreated cells (see SI, Figure S9, Bottom).
Considering the very good quality of the signal in cells, the
photostability of dye 6e was evaluated and compared to a
commercially available bodipy fluorophore, LysoTracker Green
DND-26. Interestingly, compound 6e shows a very good
explain the better fluorescence properties observed for
compound 4.
With this information in hand, the potential of this new family
of fluorophores was further investigated by considering the
influence of an extra nitrogen in the azole ring as well as the
effect of substituents on the reactivity and fluorescence
properties. Various tetrazapentalenes and triazapentalenes
bearing donor or acceptor groups on the azole moiety were
synthesized in moderate to excellent yields in accordance with
synthetic pathway A. In addition, aryl substituted
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J. Name., 2013, 00, 1-3 | 3
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photostability (Figure 4) with a remaining signal up to 480 s (see
SI for details). In this context, the cytotoxicity of probe 6e was
evaluated with the Alamar Blue assay and more than 95% of cell
viability was observed up to 170 µM concentration (see SI for
details).
Notes and references
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DOI: 10.1039/C9CC03765A
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We created an original versatile tricyclic scaffold offering highly
promising fluorescent properties. The fused diazine on the
triazapentalene ring induced a strong bathochromic shift of the
emission bands combined with improved quantum yield values
due to a probable ICT process. This family appears to be non-
toxic, highly photostable, chemically stable and some
compounds are amphiphilic. With epifluorescence microscopy
experiments we demonstrated that compound 6e can serve as
a fluorescent probe for the optical imaging of living cells. A
combination of small size/low molecular weight, good
photostability, enhanced photophysical properties, large Stokes
shifts, and versatility makes the reported fused triazapentalene
family of fluorophores
a promising tool for biological
applications and is currently being explored for optical imaging.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgment
We thank the master students- Emeline Cristofaro and Dorian
Guillot, for preliminary theoretical calculations of some
compounds. This work was supported by the French ministry of
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(ANR-11-LABX-0018-01), University of Orléans and Région
Centre Val de Loire. S.P. acknowledges support from Institut
National de la Santꢀ et de la Recherche Mꢀdicale (INSERM). In
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Cancer, La Région Centre and the Réseau Canaux Ioniques du
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