Sydnone-coumarins as clickable turn-on fluorescent sensors for molecular imaging

Article abstract of DOI:10.1039/c8cc06070c

Copper-catalyzed and copper-free sydnone-alkyne cycloaddition reactions have emerged as complementary click tools for chemical biology but their use in bioorthogonal labeling is still in its infancy. Herein, combinations of alkynes and coumarin-sydnones were screened for their ability to generate pyrazole products displaying strong fluoroscence enhancement compared to reactants. One sydnone was identified as a particularly suitable new turn-on probe for protein labeling.

Full text of DOI:10.1039/c8cc06070c

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Sydnone-Coumarins as Clickable Turn-On Fluorescent Sensors for
Molecular Imaging
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
b
b
Elodie Decuypère, Margaux Riomet, Antoine Sallustrau, Sarah Bregant, Robert Thai, Grégory
a
c
a
a
Pieters, Gilles Clavier, Davide Audisio and Frédéric Taran*
DOI: 10.1039/x0xx00000x
Copper-catalyzed and copper-free sydnone-alkyne cycloaddition reactions have emerged as complementary click tools for
chemical biology but their use in bioorthogonal labeling is still in its infancy. Herein, combinations of alkynes and
coumarin-sydnones were screened for their ability to generate pyrazole products displaying strong fluoroscence
enhancement compared to reactants. One sydnone was identified as particularly suitable new turn-on probe for protein
labeling.
www.rsc.org/
1
Clickable fluorogenic probes are important chemical tools to azides. Very recently, sydnone-based luminophores were
0
1
label, visualize and study biomolecules. They are typically developed and used for the labelling of proteins and living cells
1
designed with a profluorophore bearing a clickable function using SPSAC. An example of fluorogenic photoligation
1
that both suppresses the fluorescence and allows the ligation reaction using diarylsydnones was also recently disclosed and
12
2
to the corresponding click partner. The structural changes applied to protein labeling.
accompanying the click reaction lead to unquenching of the In this contribution, we designed a series of sydnone-
profluorophore and result in the enhancement of the coumarins and evaluated their ability to behave as effective
fluorescence. Many of these probes are based on the Cu(I)- fluorogenic clickable turn-on probes, both in CuSAC and SPSAC
catalyzed Azide-Alkyne [3+2] Cycloaddition reaction (CuAAC), reactions. The enhancement of the fluorescence results from
which can be considered as the archetype of click chemistry, the suppression of internal quenching and the structural
3
and its copper free version using strained cycloalkynes. Over modifications triggered by the transformations (Scheme 1).
5
4
series of azides and alkynes
the last decade,
a
R'
R'
profluorophores have been developed and successfully used
for biological imaging. Inspired by this remarkable amount of
work, we wondered if sydnones might be used to expand the
toolbox of clickable fluorogenic probes. Our group identified
sydnones as interesting dipole partner for the Cu-catalyzed
cycloaddition reaction with terminal alkynes, leading to 1,4-
pyrazoles. This reaction was called CuSAC for Cu-catalyzed
[Cu]
fluorophore
N
CuSAC
N
O
profluorophore
N
O
N
R'
6
Sydnone Alkyne Cycloaddition. Following this work, the
fluorophore
N
R'
copper free version (SPSAC, Strained Promoted Sydnone
SPSAC
N
Alkyne Cycloaddition) was developed by the group of J. W.
7
8
9
Chin, our group and by the group of J. M. Murphy. Later, we
showed that sydnones can be tuned to provide highly reactive
clickable reagents for both SPSAC and CuSAC reactions,
displaying higher kinetic properties than reactions involving
Scheme 1 Schematic principle of turn-on sydnones probes.
Coumarins were chosen as profluorophore candidates because they
are stable, small in size and routinely used for molecular imaging.
Substitutions in position 3- and 7- of coumarin dyes are well known
to have a strong impact on their fluorescence properties. We
reasoned that the mesoionic ring could be installed on these
positions and synthetized a series of coumarin derivatives, as
depicted in Scheme 2.
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Journal Name
Sydnone building blocks
O
O
a)
2
CO H
O
O
O
O
O
O
O
O
NH 1) tBuONO
) TFAA
O
O
R
N
N
O
O
R
NH
2
CHOCO
2
H
2
NaBH CN
3
O
O
N
O
O
N
N
N
N
N
O
R
3
, 70%
O
R = Me, 30%
R = CF
1 R = Me, 97%
2 R = CF
CH3 1, 97%
CF3 2, 51%
H
3
CO
O
O
3
, 78%
3
, 51%
O
O
HO
O
O
O
OH
N
N
O
b)
CO
2
H
R
N
N
6, 62%
1
) NaNO
2
,
O
O
O
O
HO
2
C
HCl (47%)
CHO
N
O
5, 68%
N
R
4
, 78%
O
N
N
O
O
O
NH
N
N
O
2
HO C
2) Ac
(
2
O
O
HATU, TEA
or
N
O
O
56%)
O
O
2
N
N
N
1
3, 28%
N
O
O
O
9
, 21%
1
) TMSCHN
2
O
3
-12, 21-85%
N
N
2
) Piperidine, AcOH
O
O
O
N
N
O
O
8, 84%
O
O
Scheme 2 Synthetic routes to 3- and 7-sydnone-coumarins.
7, 85%
O
O
O
F
N
N
O
O
Though the synthesis of coumarins is well established, and the
insertion of a variety of heterocycles has been reported, sydnone-
Br
N
N
12, 78%
O
O
2
EtO C
N
N
O
11, 66%
O
1
3
1
0, 49%
coumarin scaffolds are still unexplored. 7-sydnone-coumarins 1
and 2 were obtained from commercially available 7-amino-
coumarins through a standard reductive amination step in presence
of glyoxylic acid and sodium cyanoborohydride as a reducing agent.
The glycine intermediates were nitrosylated in presence of tert-
butyl nitrite, and subsequent cyclodehydration in presence of
trifluoroacetic anhydride delivered sydnones 1 and 2 in 97 and 51%
yield. Looking for a more efficient approach, we reasoned that 3-
sydnone-coumarin derivatives might be assembled at the late-stage
of the process, thus enabling a divergent access to a larger variety
Alkyne building blocks
MeO
C
B
A
OH
H
2
CO H
O
N
2
HO C
N
H
4
D
E
N
O
F
G
H
I
O
Scheme 3 Sydnones and alkynes building blocks used in this work.
of molecules. We identified sydnone 13 as the key intermediate to Combination of reaction partners were carried out in 96-wells
build up diversity. 13 was obtained at a multigram scale from plates, leading to 108 reactions conducted overnight at 60 °C for
commercial
iminodiacetic
acid
through
a
traditional CuSAC (alkynes A-G) and at room temperature for SPSAC (alkynes H
nitrosylation/cyclodehydration protocol in 28% overall yield. The and I). Each crude was then analysed by LC/MS to evaluate the
conversion of 13 to the desired 3-sydnone coumarins 3-12 was conversion of the reaction, directly screened for fluorescence
achieved by means of two protocols. Esterification in presence of enhancement in 380-600 nm emission range and subsequently
salicylic aldehydes and HATU followed by
a spontaneous compared with blank experiments carried out without alkyne. Most
Knovenagel cyclization allowed to assemble compounds 3, 9-11 in a pyrazole products showed distinct enhancement in fluorescent
single step in 21-70% yield. The protocol turns out to be extremely intensity regardless of the structures of both the alkynes and the
effective in the presence of electroneutral and electron-poor sydnones (Figure 1).
substituents on the aldehyde. Unfortunately, with electron-rich
substituents, yields dropped significantly. To overcome this lack of
reactivity, an alternative procedure was developed: methylation of
the carboxylic acid in presence of trimethylsilyldiazomethane
delivered the corresponding methyl ester in 91% yield. Treatment
of this ester with piperidine and salicyl aldehydes in n-butanol
delivered 4-8 and 12 in 51-85% yield. With a library of 12 sydnone-
coumarins reactants in our hands, we next explored their reactivity
towards seven terminal alkynes A-G and two cycloctynes H and I
(
Scheme 3).
Figure 1 Screening results. The color code indicates the
emission region of the pyrazole product. Number inside square
corresponds to the ratio of fluorescence intensities (FI)
between the pyrazole products and their corresponding
sydnones. BPDS: Bathophenanthroline-disulfonate; Na. Asc.:
sodium ascorbate; Excitation wavelength: 365 nm.
With the exception of three reactions, which failed due to
solubility issues, all other combinations were completed after
one night as monitored by LC/MS. 24 alkyne/sydnone
combinations induced a more than 20 fold increase of the
2
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DOI: 10.1039/C8CC06070C
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fluorescence signal upon click reaction. Most of these Figure 3 Left: ground state (top) and first excited state
examples were reproduced in larger scale, the corresponding
pyrazoles isolated and their optical properties as well as their
kinetic constant carefully determined, confirming the results
obtained during the screening (see supporting information).
(
bottom) optimized geometries for sydnone 8 (θ refers to the
dihedral angle between coumarin and sydnone). Middle:
energy diagram of the calculated transitions with their
oscillator strength values (f). S0 column refers to calculations
carried out using the ground state optimized geometry and S1
to calculations carried out using the first excited state
Globally, alkyne
and the most interesting sydnone partners both in term of
fluorescence enhancement and Stokes shift value.
comparative study between CuSAC and SPSAC was then
carried out using the combination and respectively.
The pyrazoles were first prepared, isolated and characterized
D was found the best terminal alkyne and 5, 7
8
A
optimized geometry. Right: main orbitals involved in the S
and S /T transitions.
1 1
/T
8/
D
8/I
2
2
to determine quantum yields. The kinetic of the click reactions The first excited state is a charge transfer one from the
at room temperature were then studied by fluorescence electron rich amino coumarin to the sydnone with a large
monitoring and confirmed by HPLC to determine the speed of
oscillator strength while the second one involves the sydnone
the reactions. The results (Figure 2) clearly highlighted SPSAC
and the lactone ring of the coumarin. In the relaxed first
as a superior tool in terms of both efficiency and rapidity.
1
excited state, two triplet states lie below S . The energy gap
Combination 8/I conducted to the quantitative formation of
pyrazole Pyr2 with a kinetic constant of k = 1.16 M .sec . This
pyrazole displays interesting fluorescent properties with a
-1
-1
between S1 and T2 is 0.9 eV and intersystem crossing is
possible and could be the main non-radiative deactivation
quantum yield of 0.57 and
enhancement as compared to sydnone
a
131-fold fluorescence pathway that inhibit the fluorescence of 8.
8
.
On the other hand, the pyrazole product (Figure S14 in SI) has
almost no geometrical reorganization in the first excited state
compared to the ground state geometry. The first transition is
an intense π -π* involving orbitals delocalized on the entire
molecule and only the T
possibility of intersystem crossing. This situation can explain
the intense fluorescence observed for Pyr1 and Pyr2
1
state is lower in energy limiting the
3
2
2
1
1
5
0
,0E+07
,5E+07
,0E+07
,5E+07
,0E+07
,0E+06
,0E+00
3,0E+07
2,5E+07
2,0E+07
1,5E+07
1,0E+07
5,0E+06
0,0E+00
0 min
15 min
1 h
0
min
15 min
1 h
3
1
0 min
.5 h
30 min
.
2 h
1
2
.5 h
.5 h
2 h
2.5 h
3.5 h
3.5 h
The ability of probe
8 to label proteins was evaluated via
CuSAC and SPSAC on Myoglobin (MYO) previously modified by
a terminal alkyne or by the cyclic alkyne
information for protein preparation).
I (see supporting
4
50
550
Wavelength(nm)
650
450
500
550 600
Wavelength(nm)
650
700
Figure 2 Comparative kinetic and photo-physical properties of
sydnones for fluorogenic CuSAC and SPSAC reactions,
solvent: PBS buffer containing 1% DMF. CuSAC conditions: [
1 µM; [ ] = 100 µM; [CuSO . 5H O] = 50 µM; [BPDS] = 300
µM; [Na. Asc.] = 2.5 mM. SPSAC conditions: [
00 µM.
8
8
]
=
D
4
2
8
] = 1 µM; [I] =
1
To understand the origins of such fluorescence enhancement,
TDDFT calculations have been carried out on sydnone and
8
corresponding pyrazole at the M06-2X/6-311+g(d,p) level of
theory using the IEF version of the PCM solvation model of
water to take into account solvent effects. TDDFT have been
done on molecules in their optimized ground state geometries
as well as their first excited state optimized one. In the ground
state, the coumarin and sydnone are twisted by an angle of
28° but compound
8 tends to planarity in its first excited state
(Figure 3).
Figure 4 a) SPSAC reaction for protein labeling using the turn-
on coumarin sydnone 5, 7 or
8. b) Comparison of ESI-MS
deconvoluted spectra of MYO-
I with Myoglobin labelled by
probe
triangles to MYO-
n = 2), orange triangles (n = 1) and orange double triangles (n
2) to -derived Myoglobin; green triangles (n = 1) and green
double triangles (n = 2) to -derived Myoglobin; blue triangles
n = 1) and blue double triangles (n = 2) to -derived
5,
7
or
8
. Grey squares refer to unmodified MYO, black
I
(n = 1) and black double triangles to MYO-
I
(
=
5
7
(
8
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Myoglobin. c) SDS-PAGE analysis of the labeling of MYO-
Journal Name
I
by
8
3
For reviews see for example: a) E. M. Sletten, C. R. Bertozzi,
Acc. Chem. Res. 2011, 44, 666; b) P. Thirumurugan, D.
Matosiuk, K. Jozwiak, Chem. Rev. 2013, 113, 4905; c) M. van
Dijk, D. T. S. Rijkers, R. M. J. Liskamp, C. F. van Nostrum, W.
E. Hennink, Bioconjugate Chem. 2009, 20, 2001.
in buffer (lane I) and in HeLa lysate (lane II). Lane III
corresponds to HeLa lysate only. The left and right panels
correspond to fluorescence and silver staining respectively.
4
5
6
See for example: a) P. Shieh, M. J. Hangauer, C. R. Bertozzi, J.
Am. Chem. Soc. 2012, 134, 17428; b) P. Shieh, V. T. Dien, B. J.
Beahm, J. M. Castellano, T. Wyss-Coray, C. R. Bertozzi, J. Am.
Chem. Soc. 2015, 137, 7145.
See for example: a) J. Qi, M-S. Han, Y-C. Chang, C-H. Tung,
Bioconjugate Chem. 2011, 22, 1758; b) J. C. Jewett, C. R.
Bertozzi, Org. Lett. 2011, 13, 5937; c) F. Friscourt, C. J. Fahrni,
G-J. Boons, J. Am. Chem. Soc. 2012, 134, 18809.
In contrast to CuSAC that failed to provide efficient fluorescent
labeling (Figure S9), SPSAC gave very promising results. For the
investigation of the fluorescent labeling using SPSAC, sydnones
5
,
7
and
analysis (Figure 4b), reaction of MYO-
sydnones led to complete conversion of MYO-
8
were compared (Figure 4a). According to mass
with each of these
into pyrazole
I
I
a) S. Kolodych, E. Rasolofonjatovo, M. Chaumontet, M-C.
Nevers, C. Créminon, F. Taran, Angew. Chem. Int. Ed. 2013
adducts in a quantitative manner. The resulting fluorescent
proteins were easily detectable using conventional imager for
,
2, 12056. For recent reviews on the chemistry and reactivity
5
the case of
7
and
8
myoglobin adducts. The most sensitive
for an emission collection
of sydnones, see: b) D. L. Browne, J. P. A. Harrity,
Tetrahedron, 2010, 66, 553; c) E. Decuypère, L. Plougastel, D.
Audisio, F. Taran, Chem Commun. 2017, 53, 11515.
S. Wallace, J. W. Chin, Chem Sci. 2014, 5, 1742.
L. Plougastel, O. Koniev, S. Specklin, E. Decuypere, C.
Créminon, D-A. Buisson, A. Wagner, S. Kolodych, F. Taran.
Chem. Commun. 2014, 50, 9376.
M-K. Narayanam, Y. Liang, K. N. Houk, J. M. Murphy, Chem.
Sci. 2016, 7, 1257.
0 H. Liu, D. Audisio, L. Plougastel, E. Decuypere, D-A. Buisson,
O. Koniev, S. Kolodych, A. Wagner, M. Elhabiri, A.
Krzyczmonik, S. Forsback, O. Solin, V. Gouverneur and F.
Taran. Angew. Chem. Int. Ed. 2016, 55, 12073.
detection was obtained with probe
8
at 515 nm (see supporting information, S12).
7
8
We then controlled the selectivity of the SPSAC reaction by
labeling MYO-I with 8 in a HeLa (human epithelial carcinoma
cell line) whole cell lysate (Figure 4c). In this complex media,
the fluorogenic click reaction was also successful and formed
the fluorescent protein adduct (Lanes I and II, left panel)
orthogonally to the other proteins of the lysate that remained
unlabelled (lane III, left panel).
9
1
1
1
1
1 L. C-C. Lee, H. M-H. Cheung, H-W. Liu, K. Kam-Wing. Chem.
Eur. J. 2018, DOI: 10.1002/chem.201803452.
2 L. Zhang, X. Zhang, Z. Yao, S. Jiang, J. Deng, B. Li, Z. Yu, J. Am.
Chem. Soc. 2018, 140, 390.
3 During the preparation of this manuscript, Friscourt et al.
published a coumarin-sydnone compound displaying turn-on
properties: C. Favre, F. Friscourt, Org. Lett. 2018, DOI:
Conclusions
We have herein reported the synthesis of a series of coumarin-
based sydnones, using a new and efficient synthetic route.
These compounds have then been combined with several
alkynes leading to the formation of more than 100 pyrazoles
products whose fluorescence properties were systematically
compared to the starting sydnones. This combinatorial
approach led to the identification of more than 20 systems
displaying interesting turn-on fluorescence properties and
highlighted 3-sydnone-coumarins with electron-donating
groups at the 7-position as suitable turn-on fluorogenic
1
0.1021/acs.orglett.8b01587.
probes. Among them, sydnone
8 was particularly studied and
proved to be a valuable tool for the labeling of proteins even in
complex biological media. This study also clearly indicates the
superior efficiency of the strained-promoted reaction SPSAC
over the Cu-catalysed one, CuSAC, for fluorescent labeling of
biomolecules.
Acknowledgements
This work was supported by CEA. The authors thank Elodie
Marcon, David-Alexandre Buisson (DRF-JOLIOT-SCBM, CEA) for
the analytical support and Dr. Livia Tepshi (DRF-JOLIOT-
SIMOPRO, CEA) for kindly providing us HeLa whole cell lysate.
References
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a) W. Xu, Z. Zeng, J-H. Jiang, Y-T. Chang, L. Yuan, Angew.
Chem. Int. Ed. 2016, 55, 13658; b) C. Le Droumaguet, C.
Wang, Q. Wang, Chem. Soc. Rev., 2010, 39, 1233.
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M. E. Jun, B. Roy, K. H. Ahn, Chem. Commun. 2011, 47, 7583.
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Table of content
Sydnone-Coumarines as Clickable Turn-On
Fluorescent Sensors for Molecular Imaging.
Elodie Decuypere, Margaux Riomet, Antoine
Sallustrau, Sarah Bregant, Robert Thai, Gregory
Pieters, Gilles Clavier, Davide Audisio and Frédéric
Taran*
Sydnone-coumarines compounds are interesting
turn-on fluorogenic probes for protein labeling.