Two-Photon Absorption and Cell Imaging of Fluorene-Functionalized Epicocconone Analogues

Article abstract of DOI:10.1002/chem.201902731

Epicocconone 1 is a natural chromophore isolated from the fungus Epicoccum nigrum that has shown applications in proteomics and fluorescent microscopy thanks to its unique pro-fluorescence properties. The modification of the skeleton of the natural produc

Full text of DOI:10.1002/chem.201902731

A Journal of
Accepted Article
Title: Two-photon absorption and cell imaging of fluorene-
functionalized epicocconone analogues
Authors: Samuel Oger, Damien Schapman, Romain Mougeot,
Stephane Leleu, Noelle Lascoux, Patrice Baldeck, Magalie
Benard, Thibault Gallavardin, Ludovic Galas, and Xavier
Franck
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To be cited as: Chem. Eur. J. 10.1002/chem.201902731
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10.1002/chem.201902731
Chemistry - A European Journal
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Two-photon absorption and cell imaging of fluorene-
functionalized epicocconone analogues
Samuel Oger,[a] Damien Schapman,[b] Romain Mougeot,[a] Stéphane Leleu,[a] Noëlle Lascoux,[c] Patrice
Baldeck,[c] Magalie Bénard,[b] Thibault Gallavardin,[a] Ludovic Galas,[b] Xavier Franck*[a]
Abstract: Epicocconone 1 is a natural chromophore isolated from
the fungus Epicoccum nigrum that has shown applications in
proteomics and fluorescent microscopy thanks to its unique pro-
fluorescence properties. The modification of the skeleton of the
natural product by replacing the triene side chain by a fluorenyl
scaffold can noticeably increase the fluorophore’s absorption
coefficient. The synthesis of the analogues of the natural product
has been made possible by the use of a palladium catalyzed
carbonylation reaction, allowing the construction of the β-keto-
dioxinone key intermediate. Two-photon absorption cross section
measurements of the fluorenyl epicocconone analogues show a
structure-dependency with values ranging from 60 GM to 280 GM
and live cell imaging show intense staining of intracellular vesicles-
like structures around the nucleus.
aromatic ring, and particularly a naphthalene ring, which led to
the improvement of the spectroscopic properties giving rise to
the more photostable naphtyl derivative 2 (Figure 1).7 We
recently reported that 2 could be used for live cell imaging,
showing higher brightness compared to epicocconone 1 as well
as ability to be excited by a pulsed laser, although its skeleton
was not designed for that.8
Two-photon excited (2PE) fluorescence microscopy is a more
and more prized technique to study living systems. Like classical
confocal fluorescence imaging, it enables to temporally follow
living processes at the sub-cellular scale, and to discriminate
specific biomolecules thanks to fluorescent tags. In addition, it
also provides less out of focus photodamage, and it is less
impacted by diffusion in biological tissues allowing obtaining
deeper 3 dimensional images.9-11 However, few optimized two-
photon absorbing fluorophores are commercially available and
very few offer functionalities such as epicocconone 1. Generally,
the design of two-photon probes for biological applications have
to cope with the contradiction between biocompatibility and two-
photon absorption efficiency which requires extended π -
Introduction
conjugation.12,13
Epicocconone 1 (Figure 1) is a natural chromophore isolated
from the fungus Epicoccum nigrum drawing significant interest in
proteomics and fluorescent microscopy thanks to its unique pro-
fluorescence properties.1,2 Indeed, the reaction of epicocconone
with protein nucleophilic lysines leads to the immediate
formation of an enamine featuring near infrared absorption and
fluorescence emission, making it suitable for the detection of
proteins. One of its principal assets is that this conjugation
reaction can be reversed allowing releasing very conveniently
the protein from the fluorophore, facilitating mass spectrometry
analyses.3-5
R
O
O
HN
lysines
primary amines
O
O
O
OH
O
O
O
O
OH
OH
OH
OH
epicocconone 1, Native form
Enamine form
fluorescent
photosensitive
photosensitive
ref. 7b
O
O
O
O
O
O
O
O
O
this work
R1
R1
OH
R1 = n-Bu, CH2CH2OMe
R2 = H, p-OMePh, p-Ph2NPh
OH
O
R2
On the other hand, it has been shown that epicocconone 1 was
suffering from photosensitivity and that the triene tail was
responsible for non-radiative deexcitation pathways.6 A solution
brought to these issues was to replace the triene moiety by an
fluorenyl synthetic analogues 24,25
naphtyl synthetic analogue 2
photostable
designed for two-photon imaging
Figure 1: epicocconone 1, naphtyl analogue 2 and fluorene derivatives
In this work, the optimization of the epicocconone skeleton
addresses two-photon absorption (2PA) maximum wavelength,
2PA cross section, and fluorescence switching between a low
fluorescent native form and a highly fluorescent enamine (Figure
1). Our benchmark was the previously described naphthyl
derivative 2, featuring a sharp 2PE maximum in both forms very
relevant to allow multiplexing in two-photon microscopy.8
Nevertheless, it can be improved by introduction of polarizable
functions such as alkoxy or amino groups. In order to retain the
functional core responsible for pro-fluorescence, only the
naphthalene ring was modified and replaced by a fluorene ring.
This moiety was chosen because many fluorene derivatives
feature noticeable “figure of merit” i.e. ratio between two-photon
[a]
Dr. S. Oger, Dr. R. Mougeot, Dr. S. Leleu, Dr. T. Gallavardin, Dr. X.
Franck
Normandie Univ, CNRS, INSA Rouen, UNIROUEN, COBRA (UMR
6014 and FR 3038), 76000 Rouen, France
E-mail: xavier.franck@insa-rouen.fr
[b]
[c]
D. Schapman, Dr. M. Bénard, Dr. L. Galas
Normandie Univ, Inserm, UNIROUEN, PRIMACEN, Cell Imaging
Platform of Normandy, Institute for Research and Innovation in
Biomedicine (IRIB), 76000 Rouen, France
Dr. N. Lascoux, Dr. P. Baldec
Ecole Normale Supérieure de Lyon, CNRS, Université Lyon
1, Laboratoire de Chimie UMR 5182, 46, allée d’Italie, F-
69364, Lyon Cedex 07, France
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201902731
Chemistry - A European Journal
FULL PAPER
absorption and molecular weight.14-16 Indeed, similarly to
biphenyl, fluorene scaffold extends the electron delocalization
while being of modest size, which should favor cell
internalization. Moreover, the use of a fluorene moiety rather
than biphenyl should allow shifting 2PA maximum to a slightly
higher wavelength17,18 while providing two easily functionalizable
positions.
bromofluorene 6 was converted into its iodo-bromo derivative 13
which was di-butylated as before to give 14 with 74% yield. The
bromo-iodo-fluorene 14 was then engaged in the palladium-
catalyzed carbonylation reaction, with in-situ CO generation and
in the presence of dioxinone 10, to give the brominated β-keto-
dioxinone 15 with a satisfying 80% yield. The remaining bromine
atom could then be engaged in a Suzuki coupling to introduce
aromatic substituents; however, owing to the thermal sensitivity
of the dioxinone moiety, room temperature conditions were
selected.20 The reaction of pOMePhB(OH)2 16 in the presence
of Pd(OAc)2 and K2CO3 in a EtOH/H2O mixture proved to be
very efficient with a complete conversion of 15 and a 80%
isolated yield in β-keto-dioxinone 4b. Applying the same reaction
conditions to pPh2NPhB(OH)2 17 yielded irreproducible yields in
4c; however, after a brief survey of different catalysts, the use of
Pd(SPhos) with K3PO4 in THF/H2O21 allowed us to obtain a
moderate but reproducible 43% yield in 4c.
Results and Discussion
The retrosynthesis of the so-called “fluorenyl analogues”
24-25 emphasizes the fluorene-functionalized dioxinones 4,5
and the key bicyclic keto-alcohol 3, as shown scheme 1. The β-
keto-dioxinones 4a-c and 5a-c will bear different R1 (n-C4H9,
CH2CH2OMe ) and R2 (H, pPhOMe, pPhNPh2) groups in order to
study their influence on fluorescence and solubility. Bicyclic
Bu
Bu
Bu
Bu
keto-alcohol 3 will be prepared as already reported by us.7b
n-BuLi
CO2
1) Br2
COOH
Br
Br
Br
Br
O
2) n-BuBr, tBuOK
≈
10%
O
6
11
12
O
HO
O
O
O
O
O
bicyclic keto-alcohol 3
Bu
Bu
R1
R1
t-BuOK
n-BuBr
R1 R1
OH
O
O
O
I
I2, NaIO3, H2SO4
Br
I
Br
R2
O
AcOH/H2O 20/1
90°C, 3h
70%
THF, 0°C -> RT
74%
R2
14
13
fluorenyl synthetic analogues 24a-c, 25a-c
functionalized β-keto-dioxinone
O
O
R1 = n-Bu, CH2CH2OMe
R2 = H, p-OMePh, p-Ph2NPh
10
4a: R1=Bu, R2=H
4b: R1=Bu, R2=pOMePh
4c: R1=Bu, R2=pPh2NPh
5a: R1=CH2CH2OMe, R2=H
5b: R1=CH2CH2OMe, R2=pOMePh
5d: R1=CH2CH2OMe, R2=pPh2NPh
O
80%
LiHMDS, CO
Pd(dba)2, Xantphos
Bu
Bu
Bu
Bu
O
O
O
MeO
pOMePhB(OH)2 16
Pd(OAc)2, K2CO3
EtOH/H2O (1/1)
O
O
Scheme 1: retrosynthetic scheme of fluorenyl epicocconone analogues 24-25
Bu
Bu
O
O
O
4b, 80%
Br
or
O
O
O
O
Ph2NPhB(OH)2 17
Pd(SPhos), K3PO4
THF/H2O (4/1)
15
Ph2N
The synthesis of the simple dibutyl dioxinone 4a (scheme 2)
started from commercially available 2-bromofluorene 6 which
was di-butylated by treatment with n-BuBr in the presence of t-
4c, 43%
Scheme 3: Synthesis of 4b and 4c
BuOK (R1=Bu). The so-obtained bromo derivative
transformed into the corresponding carboxylic acid
7
was
by
8
metallation with n-BuLi followed by addition of CO2. The
carboxylic acid function was then converted to the benzotriazole
derivative 9 which reacted with the lithium enolate of the simple
dioxinone 10. The resulting β-keto-dioxinone 4a was then
obtained in 4 steps from 2-bromofluorene 6 with 12% overall
yield.
To increase the water solubility of the final products, we
changed the all-carbon dibutyl R1 substituents on 4a-c by 2-
methoxyethyl residues (scheme 4). For that, using
MeOCH2CH2OTs, the functionalization of the fluorene skeleton
18 was performed, yielding the disubstituted fluorene 19 with
60% yield. At this stage, mono-iodation to 20 could be
performed, followed by a palladium-catalyzed carbonylation,
introducing the dioxinone moiety and giving the β-keto-dioxinone
5a with 44% yield. To access more substituted fluorene
derivatives, bromination followed by iodination were performed
on fluorene 19, yielding the di-halogeno fluorene 22. Iodine
selective palladium catalyzed carbonylative coupling and
introduction of the dioxinone yielded the bromo-β-keto-dioxinone
23 with a moderate 40% yield. The following Suzuki couplings
Bu
Bu
Bu
Bu
tBuOK
n-BuBr
n-BuLi
CO2
Br
COOH
R2
H
H
Br
68%
0°C -> RT
74%
8
6
7
R2=H
BtzH, SOCl2
42%
O
O
Bu
Bu
Bu
Bu
O
O
O
O
10
O
H
H
Btz
O
LDA, THF, -78°C -> RT
56%
4a
9
were performed at 40°C with pOMePhB(OH)2 16
and
Scheme 2: Synthesis of 4a
pPh2NPhB(OH)2 17 in the presence of Pd(SPhos)/K3PO4,
yielding β-keto-dioxinones 5b (62%) and 5c (41%), respectively.
It is worth noting that the yields obtained for the preparation of
dioxinones substituted by CH2CH2OMe were usually lower than
those obtained for butyl-substituted side chain; this could be
possibly explained by the metal coordination ability of the
methoxy residues.
To prepare more substituted dibutyl dioxinones with R2≠H, a
second halogen had to be introduced on the fluorene ring to
allow late functionalization. However, all attempts to apply the
same strategy as before from the dibrominated fluorene 11
proved to be low yielding in carboxylic acid 12, preventing
further synthesis (scheme 3). We then considered a recent and
elegant procedure developed by Skrydstrup,19 allowing the direct
and selective conversion of an iodoaryl derivative into a β-keto-
dioxinone by a palladium-catalyzed carbonylation. Therefore the
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10.1002/chem.201902731
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when more extended electron-donating groups were added
(Table1, Figure 2). However, Native 25b(N) and 25c(N)
emissions were very low in CH2Cl2 with quantum yields of 0.01.
Enamines (E) were then formed by addition of butylamine,
leading to an immediate striking color switching from yellow to
red; the fluorescence of 2(E) and 25a(E) noticeably increased
from around 0.25 (native form) to 0.60; more surprisingly, 25b(E)
was found to be fluorescent with a quantum yield of 0.63, while
on the other side, 25c(E) still presented very low fluorescence
with quantum yield of 0.03. The shapes of enamines spectra
were unexpectedly very similar for all compounds, the lowest
energy absorption and the emission bands superimposed
perfectly as if the fluorenyl scaffold was not directly involved in
these transitions, only modulating fluorescence efficiency.
O
O
MeO
OMe
MeO
OMe
O
10
O
O
O
NIS/TFA
18
Pd(dba)2, Xantphos
LiHMDS, CO
44%
MeCN, 80°C, 4h
I
H
O
tBuOK
MeOCH2CHOTs
59%
60%
MeO
5a
20
THF, 0°C -> RT
MeO
OMe
MeO
OMe
OMe
NBS
DMF, 60°C, 20h
I2/KIO3
I
Br
Br
50%
AcOH/H2O/H2SO4
(10/5/1)
19
22
21
74%
LiHMDS, CO
O
O
Pd(dba)2, Xantphos
40%
MeO
OMe
O
10
O
O
O
MeO
OMe
MeO
pOMePhB(OH)2 16
O
or
O
O
O
5b, 62%
Ph2NPhB(OH)2 17
Br
O
Pd(SPhos), K3PO4
THF/H2O (4/1)
MeO
OMe
23
O
O
O
Ph2N
O
5c, 41%
O
O
HN
Scheme 4: Synthesis of 5a-c
O
O
O
OH
O
O
O
O
BuNH2
Ar
OH
Ar
OH
The completion of the synthesis of the fluorenyl epicocconone
analogues was realized by condensation of dioxinones 4 and 5
with the bicyclic keto-alcohol 3, via thermal fragmentation of
dioxinones to an acylketene, following our reported procedure
(scheme 5).7b Then heating a mixture of dioxinones 4a-c or 5a-c
and ketoalcohol 3 in toluene for 30 minutes followed by addition
of triethylamine gave a series of linear lactones 24a-c and 25a-c,
albeit in much lower yields than usually obtained with dioxinones
Native form (N)
Enamine form (E)
Ar= fluorenyl: 24(N), 25(N)
Ar= naphtyl: 2(N)
Ar= fluorenyl: 24(E), 25(E)
Ar= naphtyl: 2(E)
substituted by simple aromatic rings.7
O
O
O
O
R1
R1
OH
O
H
R1= Bu: 24a (8%)
R1=CH2CH2OMe: 25a (9%)
O
O
O
O
O
O
HO
Figure 2 : Formation of enamine compounds by addition of butylamine to
native derivatives. Absorption and emission of 2 (green), 25a (black), 25b
(red) and 25c (blue) in dichloromethane : native form (N) left and enamine
form (E) right.
3
O
R1
R1
4a-c or 5a-c
OH
toluene, reflux
then Et3
O
N
MeO
R1= Bu: 24b (11%)
R1=CH2CH2OMe: 25b (4%)
O
O
O
O
To get more insight into their spectroscopic properties,
compounds were then studied in less polar solvent toluene.
R1
R1
OH
O
Ph2N
R1= Bu: 24c (10%)
R1=CH2CH2OMe: 25c (5%)
Scheme 5: Synthesis of 24a-c and 25a-c
The optical properties of fluorenyl analogues were studied and
compared with those of the parent compound in
2
dichloromethane. As 24 and 25 display almost superimposable
optical properties, only one-photon absorption/emission spectra
for 25 will be detailed in Fig. 2-3 (see Fig. S1-S4 for spectra for
24 or Table 1 for full data). Native form of 25a named 25a(N)
featured absorption and fluorescence spectra as well as
quantum yield very close to those of the native form of 2 named
2(N). As expected a small red shift was observed in absorption
Figure 3 : Absorption and emission of 2 (green), 25a (black), 25b (red) and
25c (blue) in toluene : native form (N) left and enamine form (E) right.
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Table 1: Optical properties.
[d]
[d]
[d]
[d]
Solvent
Compound
λabs
λem
ε [e]
φF
Compound
λabs
ε[e]
λem
φF
2(N)
2(E)
420
500
531
591
29500
27200
0.25[a]
0.59[c]
CH2Cl2
24a(N)
24a(E)
425
500
531
591
33800
27000
0.24[a]
0.58[c]
25a(N)
25a(E)
425
500
27400
21400
531
591
0.26[a]
0.59[c]
24b(N)
24b(E)
433
500
541
591
44700
34900
0.01[a]
0.59[c]
25b(N)
25b(E)
432
500
40300
31000
542
591
0.01[a]
0.63[c]
24c(N)
24c(E)
436
500
543
591
38900
30900
0.02[b]
0.05[c]
25c(N)
25c(E)
436
500
34100
25800
552
591
0.01[b]
0.03[c]
2(N)
2(E)
422
491
528
574
29200
30200
0.24[a]
0.69[c]
Toluene
24a(N)
24a(E)
427
491
528
574
32900
27300
0.25[a]
0.75[c]
25a(N)
25a(E)
428
491
27400
22000
528
576
0.25[a]
0.66[c]
24b(N)
24b(E)
435
491
528
574
43600
35500
0.28[a]
0.68[c]
25b(N)
25b(E)
436
491
41100
30700
528
576
0.27[a]
0.65[c]
24c(N)
24c(E)
439
491
576
574
41700
33100
0.12 [b]
0.65[c]
25c(N)
25c(E)
438
491
34200
28300
576
576
0.07[b]
0.65[c]
[a] measured using lucifer yellow (φ=0.21 in water λexc = 420 nm);22 [b] measured in water using coumarin 153 (φ=0.38 in ethanol,λexc = 450 nm);23 [c]
measured in water using rhodamine 6G (φ=0.95 in ethanol,λexc = 500 nm)24; [d] in nm; [e] in L. mol-1. cm-1
In toluene (Table 1, Figure 3) fluorescence quantum yields of
Native derivatives were respectively 0.29, 0.25 0.28 for 2(N),
25a(N) and 25b(N), while 25c(N) remained less fluorescent
(0.12). The emission spectrum of this latter also differentiated
from the others by a large Stokes shift indicative of a much
stronger internal charge transfer (ICT) at the excited state;
unexpectedly, such a behavior was not observed with 25b(N)
which emission superimposes with 25a(N).
As observed for dichloromethane, enamines 24a-c(E) or 25a-
c(E) in toluene featured very similar absorption and emission
spectra, however enamines have a much higher fluorescence
quantum yields than their native counterpart, ranging from
0.65 to 0.75 (Table 1). Spectra in dichloromethane and in
toluene also highlighted a polarity difference between native
and enamine forms, since solvatochromism was stronger for
enamines.
The strong fluorescence drop of 24a-c or 25a-c related to
solvent polarity and electron-donor strength, might be the
result of various phenomena like modulation of the
intersystem crossing efficiency, or an effect of the keto-enol
which can induce conjugation breaking, twisting or excited
state proton transfer.25,26
Indeed, epicocconone derivatives possess aromatic carbonyl
compounds, and therefore might be able to stabilize their
excited states as triplet states, due to contributions of n-π*
states described by El-Sayed rules.27 Energy levels of n-π*
states are highly responsive to solvent polarity and
substituent effects, producing drastic variations of intersystem
crossing and thus of fluorescence quantum yields like those
observed here. Triplet state generation was assessed by
measuring the amount of singlet oxygen produced under
irradiation (Figure S5), hypothesizing quantitative singlet
oxygen generation upon triplet state consumption. In
dichloromethane, only 25a(N) slightly produced singlet
oxygen while no significant singlet oxygen productions were
measured even with low fluorescent 25b(N) and 25c(N); a
more efficient non radiative deexcitation process is then
probably responsible for the quenching of both fluorescence
and intersystem crossing. Therefore, the most probable
explanation is that the fluorescence quantum varies mostly
due to keto-enol dynamics. It was previously stated that
proton transfer in the keto-enol moiety occurs in
epicocconone derivatives, leading to more stable excited
states.6b,28 In native compounds, when electron donating
groups are added, excited states present an Internal Charge
Transfer (ICT) character, capable of evolving into twisted ICT
(TICT) in polar solvents. This phenomenon induces
a
conjugation breaking at the excited state making possible a
photoinduced electron transfer (PET) between the two sides
of the twist, leading often to fluorescence quenching.29,30
Occurrence of such TICT results in a large stokes shift as for
24c(N) in toluene, but may also be the reason for the
fluorescence quenching in more polar dichloromethane for
25b(N) and 25c(N).
In the other hand, enamine forms possess very different
spectroscopic properties, their stronger solvatochromism
effect in absorption highlights their larger excited state dipole
moment oriented from the enamine to keto-enol.6c
Unexpectedly, the fluorenyl scaffold does not seem to be
directly involved in absorption and emission transitions,
resulting in modulation of fluorescence efficiency without any
spectral change. Enamines act as if the conjugation was
broken at the level of the keto-enol both at ground state, and
excited state, since the absorption and emission bands of all
derivatives superimposed strictly. Therefore, the fluorenyl part
of the molecule acts only as a fluorescence quantum yield
modulator probably proceeding though electron transfer.
This phenomenon is very instructive on how to modulate
epicocconone derivatives to obtain
a
large gain in
fluorescence quantum yield between native and enamine
forms by adapting the oxidation potential of the aromatic
moieties.
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2PA cross sections were measured through 2PE
fluorescence in the range of 720-1050 nm using Styryl 9M
(900-1050 nm), Coumarin 510 (720-920 nm), and Coumarin
540 (720-1000 nm) as standards.31,32 Two-photon and one--
spectra were stacked to compare their respective transition
energies (Figure 4).
Figure 5 : Two-photon absorption cross section of 24a(E) (black), 24b(E)
(red), and 24c(E) (blue) in toluene.
Figure 4 : Two-photon absorption cross section (bottom and left axes)
stacked with one-photon absorption (1PA) (right and top axis) of (A) 2, (B)
25a, and (C) 25b in CH2Cl2. Native form (N) in black and enamine (E) in
red.
After having determined the photophysical characteristics of
the fluorenyl derivatives, compounds 25a-c, expected more
water-soluble than 24a-c, were engaged in two-photon cell
imaging. Staining of living PC12 cells was obtained after 45
min of incubation with dyes at a concentration of 10-6 M in
DMEM followed by a washing step and medium renewal
(solubilization of dyes was facilitated with the nonionic
surfactant polyol Pluronic® F-127, Figure S6). For two-photon
imaging of living PC12 cells, all dyes were excited at 775 nm
and fluorescence was collected from 570 to 640 nm. A pixel-
time of 600 ns and a laser power of 0.627 mW, measured
after a 25x objective (NA 0.95, water immersion), were
necessary to detect and compare fluorescent signals and to
obtain optimized images. In these conditions, fluorescence
intensity obtained for cell-labelling analogue 25a dye was
slightly (∼15%) different from that detected for analogue 2
(Fig. 6c). In contrast, fluorescent intensities in labeled cells
obtained with analogues 25b and 25c were dramatically low
(Fig. 6c). Therefore, cell labeling studies with fluorene-
functionalized epicocconone analogues were then focused on
analogue 25a. As indicated by a large view (optical zoom =
0.75), analogue 25a labeled most if not all cultured cells. In
addition, a broad cellular labeling was observed including the
cell body and processes of PC12 cells (Fig. 6a) suggesting
very good loading properties. At a higher magnification
(optical zoom = 2.5), XYZ acquisitions revealed that analogue
25a induced i) a faint staining of the plasma membrane, ii) an
intense staining of numerous intracellular vesicles-like
structures around the nucleus and iii) a moderate staining of
filamentous structures in the cytoplasm. In contrast, ZX and
ZY projections revealed that analogue 25a did not label the
nucleus of PC12 cells (Fig. 6b). These results are very similar
to those obtained in our previous study8 in which analogue 2
co-localized with the Golgi apparatus, mitochondria and
vesicle-like structures suggesting that epicocconone dyes
labels preferentially lipophilic/membrane microenvironments
in PC12 cells. However, a striking difference with analogue
25a is the absence of nuclear membrane staining that could
be observed for analogue 2. Among highly expressed single
localized nuclear membrane proteins is Lamin B1 that is
detected through immunofluorescence in PC12 cells34 and
that could be differentially revealed with analogue 2 and
In dichloromethane, 2(N), and 25a(N), featured only one
sharp 2PA band at 720-780 nm, (respectively 140 and 186
GM at 740 nm), this band does not match with any major one-
photon absorption band; 25b(N) and 25c(N) were not
fluorescent enough to provide an accurate measurement of
2PA cross section. Upon butylamine addition 2PA spectra
changed, a 20 nm red shift and a new 2PA absorption band
with an energy corresponding exactly to the 1PA transition
were observed. This energy matching between one and two-
photon absorption is indicative of
a dipolar molecule.
Interestingly, for 25b(E), only the lowest energy band is
dipolar while the other is higher in energy than the observed
1PA bond, demonstrating that there is no direct correlation
between these two bands. 2(E) and 25a(E) are quite efficient
absorbers and are suitable for 2PE fluorescence imaging by
comparison with other fluorophores.33 It is worth noting that it
is also possible to excite selectively enamine form at around
1000 nm, independently from its native form.
2PA measurements in toluene allowed obtaining spectra for
compounds containing electron donor groups. Like previously
observed, conversion to enamine adducts led to 2PA red shift
and apparition of a new low energy band matching with 1PA
(Figure 5).
As expected, the introduction of more extended electron
donor groups increased the 2PA cross section raising from 60
GM (24a(E)), 150 GM (24b(E)) to 280 GM (24c(E)) and
featuring a 50 nm red shift. However, it did not influence, the
lower energy absorption band as if the associated transition
was only localized on the unchanged epicocconone bicyclic
part of the molecule. This behavior is thoroughly in
accordance with 1PA and fluorescence properties of
enamines that did not change with electron donor
modification, suggesting a conjugation breaking in both
ground and excited state.
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10.1002/chem.201902731
Chemistry - A European Journal
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analogue 25a. In addition, effects of epicocconone analogues
were tested through fluorescent membrane integrity test with
the NucGreen reagent. A 1-hr incubation with 2, 25a and 25b
did not alter viability of PC12 cells (Fig. 6, panel d). In
contrast, the analogue 25c significantly (p<0.05) increased
the percentage of cell death. When the incubations were
prolonged to 24-hr, the epicocconone analogues had no
significant effect on cell death compare to control conditions
(Fig. 6, panel d). All together, these results suggest that the
analogue 25a presents interesting characteristics for live cell
imaging.
conjugation was broken at the keto-enol site. This may lead to
fluorescence quenching by PeT, provided that oxidizable
moieties are present. Interestingly, this phenomenon can be
modulated and will allow the development of new analogues
with increased OFF->ON behavior.
Cell staining could be performed on PC12 cells with fluorene
derivative
25a,
showing
intense
labeling
of
lipophilic/membrane microenvironments without staining of
the nuclear membrane, which is a striking difference with
analogue 2.
Experimental Section
General methods and materials
Reagents were purchased from Sigma Aldrich and Alfa Aesar and
used without purification unless specified. Spectroscopic grade
solvents: acetonitrile and DMSO were purchased from Sigma Aldrich.
Ratios provided and percentage yields are with respect to the limiting
reagent. Temperatures specified are those of the heating or cooling
bath. THF was distilled from sodium benzophenone ketyl under argon.
TLC was performed on aluminium backed silica gel 60 F254 plates
(Merck) and visualized using either a UV lamp (254 or 365 nm) or
heating with phosphomolybdic acid or potassium permanganate.
Purifications were conducted as specified with flash column
chromatography using silica gel 60 (Merck, 230-400 mesh) or with
preparative TLC plates (Merck, 1000 µm thickness). NMR spectra
were recorded on a Bruker Avance DMX-300 spectrometer and
calibrated using residual undeuterated solvent as an internal
reference (7.26 ppm and 77.16 ppm for 1H and 13C NMR in CDCl3).
HRMS were recorded either on a Thermo LTQ Orbitrap XL apparatus
equipped with an ESI source or on a LCT Premier XE bench top
orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer
(Waters Micromass) equipped with an ESI source. LRMS (ESI): data
were obtained by direct injection using a Finnigan LCQ Advantage
MAX (ion trap) apparatus equipped with an electrospray ionization
(ESI) source. Infrared (IR) spectra were recorded with a universal
ATR sampling accessory on a Perkin Elmer FT-IR Spectrum 100
spectrometer. The bond vibration frequencies are expressed in
reciprocal centimetres (cm-1).
Figure 6: Characteristics of epicocconone analogues through two-photon
fluorescence microscopy. (a) Imaging of analogue 25a-labeled PC12 cells
through two-photon (2P) microscopy (large view). (b) Subcellular staining
of living PC12 cell in 2P. Maximum intensity projection (XY) and related
orthogonal views (ZX and ZY) of analogue 25a-labeled cell. Scale bar 10
µm. (c) Comparison of fluorescence intensity obtained for cell-labeling
epicocconone dyes through 2P excitation. Each value represents the mean
(± S.E.M.) of twelve independent experiments. *p < 0.5 vs analogue 2, ***
p < 0.001 vs. analogue 2. (d) Effect of epicocconone compounds on cell
viability. Measurement of the cytotoxic effect of epicocconone analogues
(2, 25a, 25b, 25c) on PC12 cells after 1 hr or 24 hrs through fluorescent
membrane integrity test. Each value represents the mean of the
percentage of dead cell (± sem) of two independent experiments
performed on at least 600 cells for each measurement. n.s., no statistical
significance versus control; *, p<0.05, statistical significance versus control
7-hydroxy-3,3,7-trimethyl-3,4-dihydro-6H-isochromene-6,8(7H)-
dione (3) [1293991-92-0] was synthesized from methyl atratate as
previously reported.7b
2-bromo-9,9-dibutyl-9H-fluorene (7) [88223-35-2] was synthesized
from 2-bromo-9H-fluorene (6) as previously reported.35
2-bromo-7-iodo-9H-fluorene (13) [123348-27-6] was synthesized
from 2-bromo-9H-fluorene (6) as previously reported.36
2-bromo-9,9-dibutyl-7-iodo-9H-fluorene (14) [319906-46-2] was
synthesized from 2-bromo-7-iodo-9H-fluorene (13) following35 and
data were similar to those reported.37
Conclusions
We have prepared a series of epicocconone derivatives
bearing fluorenyl moieties, by synthesizing highly
functionalized dioxinones through palladium-catalyzed
carbonylation reactions. The introduction of the fluorenyl
moiety resulted in increase of the two-photon absorption
cross section of the enamine forms (E), up to 280 GM and a
50 nm red-shift of the emission when adding strong electron
donor moieties. However, it is also observed that the two
parts of the analogues, namely the bicyclic epicocconone
scaffold and fluorene moiety behave independently, as if the
6-(2-(9,9-dibutyl-9H-fluoren-2-yl)-2-oxoethyl)-2,2-dimethyl-4H-1,3-
dioxin-4-one (4a): To a solution of 1H-benzotriazol (1.77 g, 14.9
mmol) in CH2Cl2 (20 mL) was added SOCl2 (0.27 mL, 3.72 mmol).
After 30 min at RT a solution of the fluorene (8) (1.2 g, 3.72 mmol) in
CH2Cl2 (20 mL) was added. After 4 h at RT the reaction mixture was
filtered and the filter cake was rinced with a 2 M solution of NaOH (50
mL). The filtrate was extracted with CH2Cl2 (3x80 mL). The organic
phases were combined, dried over MgSO4, filtered and concentrated
in vacuo. The residue was taken up in Et2O (20 mL) and the filtrate
was concentrated in vacuo affording the product (9) (0.65 g, 1.53
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10.1002/chem.201902731
Chemistry - A European Journal
FULL PAPER
mmol, 42%) as a white solid, which was used for the next step without
any further purification.
After 18 h at RT a solution of 1 M HCl (20 mL) was added in chamber
A and the mixture was extracted with EtOAc (3x80 mL). The organic
phases were combined, dried over MgSO4, filtered and concentrated
in vacuo. Purification by flash column chromatography afforded the
corresponding brominated β-keto-dioxinone.
To a solution of HMDS (0.65 mL, 3.08 mmol) in THF (20 mL) was
added dropwise at -78 °C n-BuLi (1.23 mL, 3.08 mmol). After 30 min
at 0 °C the reaction mixture was cooled down to -78 °C and methyl
dioxinone (10) (0.57 mL, 4.62 mmol) was added. After 20 min at -
78 °C a solution of the benzotriazol derivative (9) (0.65 g, 1.54 mmol)
in THF (10 mL) was added at -78 °C. After 2 h at -78 °C the reaction
mixture was allowed to warm up at RT. After 30 min at RT a 1 M
solution of HCl (30 mL) was added and the mixture was extracted with
EtOAc (3x50 mL). The organic phases were combined, dried over
MgSO4, filtered and concentrated in vacuo. Purification by flash
column chromatography (cyclohexane/EtOAc 90/10 to 70/30) afforded
the product (4a) (0.39 g, 0.86 mmol, 56%) as a yellow oil. 1H NMR
(300 MHz, CDCl3): δ 7.93 (s, 1H, ArH), 7.90 (d, J = 7.3 Hz, 1H), 7.79-
7.76 (m, 2H), 7.42-7.36 (m, 3H), 5.46 (s, 1H), 3.98 (s, 2H), 2.08-1.96
(m, 4H), 1.73 (s, 6H), 1.13-0.97 (m, 4H), 0.65 (t, J = 7.3 Hz, 6H), 0.59-
0.48 (m). 13C NMR (76 MHz, CDCl3): δ 193.1, 165.6, 161.0, 152.2,
151.6, 147.3, 139.6, 134.6, 129.0, 128.2, 127.3, 123.3, 122.6, 121.1,
119.9, 107.5, 97.1, 55.5, 43.7, 40.1, 26.1, 25.2, 23.1, 13.9. IR (neat,
cm-1): 1710, 1686, 1606, 1453, 1376, 1264, 1210, 1087, 1046. LRMS
(ESI+, acetonitrile): m/z (%) = 447 (50) [M+H+]. HRMS (ESI): m/z
6-(2-(7-bromo-9,9-dibutyl-9H-fluoren-2-yl)-2-oxoethyl)-2,2-
dimethyl-4H-1,3-dioxin-4-one (15): Synthesized according to
General procedure
A
from compound (14). Flash column
chromatography: cyclohexane/EtOAc 90/10 to 70/30, yellow oil. Yield:
80%. 1H NMR (300 MHz, CDCl3): δ 7.91-7.89 (m, 2H), 7.72 (d, J = 8.7
Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.50-7.42 (m, 2H), 5.44 (s, 1H), 3.95
(s, 2H), 1.99-1.90 (m, 4H), 1.69 (s, 6H), 1.10-0.92 (m, 4H), 0.61 (t, J =
7.3 Hz, 6H), 0.56-0.39 (m, 4H). 13C NMR (76 MHz, CDCl3): δ 192.9,
165.5, 160.8, 154.1, 151.0, 145.8, 138.4, 134.8, 130.5, 128.3, 126.4,
123.1, 122.5, 122.3, 119.9, 107.3, 97.0, 55.6, 43.5, 39.9, 25.9, 25.0,
22.9, 13.8. IR (neat, cm-1): 1728, 1681, 1607, 1463, 1375, 1367, 1273,
1220, 1053, 1022. LRMS (ESI+, acetonitrile): m/z (%) = 547 (30)
[M(79Br)+Na+], 549 (30) [M(81Br)+Na+]. HRMS (ESI): m/z calcd. for
[C29H34O4(79Br)+] 525.1640, found 525.1646
[C29H34O4(81Br)+] 527.1620, found 527.1632.
; m/z calcd. for
6-(2-(7-bromo-9,9-bis(2-methoxyethyl)-9H-fluoren-2-yl)-2-
oxoethyl)-2,2-dimethyl-4H-1,3-dioxin-4-one (23): Synthesized
-
calcd. For [C29H33O4 ] 445.2379, found 445.2379.
according to General procedure A from compound (22). Flash
column chromatography: cyclohexane/EtOAc 70/30, yellow oil. Yield:
40%. 1H NMR (300 MHz, CDCl3): δ 8.01 (d, J = 1.0 Hz, 1H), 7.95 (dd,
J = 8.0, 1.6 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.63 (dd, J = 7.5, 4.8 Hz,
2H), 7.55 (dd, J = 8.1, 1.8 Hz, 1H), 5.45 (s, 1H), 3.96 (s, 2H), 2.99 (s,
6H), 2.76-2.60 (m, 4H), 2.40-2.29 (m, 4H), 1.74 (s, 6H). 13C NMR (76
MHz, CDCl3): δ 192.7, 165.3, 160.9, 152.6, 149.7, 145.2, 137.9, 135.2,
131.2, 128.7, 127.0, 123.5, 123.1, 122.5, 120.2, 107.5, 97.2, 68.3,
58.5, 52.0, 43.6, 39.4, 25.2. IR (neat, cm-1): 1728, 1691, 1610, 1600,
1533, 1458, 1392, 1273, 1205, 1118. LRMS (ESI+, acetonitrile): m/z
(%) = 594 (50) [M(79Br)+MeCN+Na+], 596 (50) [M(81Br)+MeCN+Na+].
HRMS (ESI): m/z calcd. for [C23H26O4(79Br)+] (M+H-acetone-CO2)
447.0994, found 447.1000.
2,7-dibromo-9,9-dibutyl-9H-fluorene (11) [188200-91-1]: To
a
solution of fluorene (18) (5.0 g, 30.1 mmol) in CHCl3 was added
dropwise at RT and in the darkness a solution of Br2 (3.6 mL, 60.2
mmol) in CHCl3. After 18 h at RT a solution of saturated Na2S2O3 (100
mL) was carefully added at 0 °C and the mixture was stirred until the
organic phase turned yellow. The resulting mixture was extracted with
CHCl3 (3x30 mL), the organic phases were combined, dried over
MgSO4, filtered and concentrated in vacuo affording the product (8.26
g, 25.5 mmol, 85%) as a pale-yellow solid which was used for the next
step without any further purification.
To a solution of crude 2,7-dibromo-9H-fluorene (2.0 g, 6.0 mmol) and
bromobutane (1.3 mL, 12.0 mmol) in THF (40 mL) was added
portionwise at 0 °C t-BuOK (2.0 g, 18.0 mmol). After 30 min at 0 °C
the mixture was allowed to warm up at RT. After 30 min at RT a
solution of 1 M HCl (40 mL) was added and the mixture was extracted
with EtOAc (3x50 mL). The organic phases were combined, dried
over MgSO4, filtered and concentrated in vacuo. Purification by flash
column chromatography (cyclohexane/EtOAc 95/5) afforded the
6-(2-(9,9-bis(2-methoxyethyl)-9H-fluoren-2-yl)-2-oxoethyl)-2,2-
dimethyl-4H-1,3-dioxin-4-one (5a): Synthesized according to
General procedure
A
from compound (20). Flash column
chromatography: cyclohexane/EtOAc 70/30, yellow oil. Yield: 44%. 1H
NMR (300 MHz, CDCl3): δ 8.02 (d, J = 1.1 Hz, 1H), 7.96 (dd, J = 8.0,
1.6 Hz, 1H), 7.79 (dd, J = 8.1, 3.5 Hz, 2H), 7.50-7.45 (m, 1H), 7.44-
7.40 (m, 2H), 5.46 (s, 1H), 3.97 (s, 2H), 2.98 (s, 6H), 2.71-2.59 (m,
4H), 2.42-2.33 (m, 4H), 1.74 (s, 6H). 13C NMR (76 MHz, CDCl3): δ
192.8, 165.4, 160.9, 150.5, 150.0, 146.4, 138.9, 134.9, 129.3, 128.6,
127.9, 123.5, 123.0, 121.3, 120.1, 107.5, 97.2, 68.4, 58.5, 51.6, 43.6,
39.6, 25.2. IR (neat, cm-1): 1724, 1682, 1635, 1605, 1450, 1390, 1374,
1270, 1199, 1109, 1014. LRMS (ESI+, acetonitrile): m/z (%) = 451
(40) [M+H+]. HRMS (ESI): m/z calcd. for [C23H27O4+] (M+H-acetone-
CO2) 367.1909, found 367.1909.
1
product (11) (1.83 g, 4.2 mmol, 70%) as an orange oil. H NMR (300
MHz, CDCl3): δ 7.55-7.50 (m, 2H), 7.45 (dd, J = 7.2, 1.7 Hz, 4H),
1.96-1.87 (m, 4H), 1.10 (dt, J = 14.8, 7.4 Hz, 4H), 0.69 (t, J = 7.3 Hz,
6H), 0.63-0.50 (m, 4H). 13C NMR (76 MHz, CDCl3): δ 152.7, 139.2,
130.3, 126.3, 121.6, 121.3, 55.8, 40.2, 26.0, 23.1, 13.0. IR (neat, cm-
1): 1597, 1568, 1448, 1414, 1397, 1130, 1108, 1057, 1005. LRMS
(ESI+, acetonitrile): m/z (%) = 459 (10) [M+Na+].
General procedure A: Synthesis of β-keto-dioxinones using
carbonylative palladium cross-coupling with double-chamber
reactor.19
General procedure B: Air-opened Suzuki-Miyaura cross-coupling
on brominated β-keto-dioxinones.
Chamber A: To a solution of methyldioxinone (10) (0.28 mL, 2.2 mmol,
1.1 equiv.) and 7-iodo-fluorene (2.0 mmol, 1 equiv.) in degassed THF
(15 mL) were added in this order at 0 °C Pd(dba)2 (0.12 g, 0.2 mmol,
0.1 equiv.), Xantphos (0.12 g, 0.2 mmol, 0.1 equiv.) and a solution of
freshly prepared LiHMDS (HMDS : 1.2 mL, 6.0 mmol; n-BuLi : 2.5 M
in hexane, 2.4 mL, 6.0 mmol) in degassed THF (5 mL). The chamber
was then sealed.
To a solution of brominated β-keto dioxinone (1 equiv.) and boronic
acid (2 equiv.) in EtOH/H2O (1:1, v/v, 0.1 M) were added K2CO3 (3
equiv.) and Pd(OAc)2 (0.1 equiv.). After 2 h at RT a 1 M solution of
HCl (10 mL) was added and the mixture was extracted with EtOAc
(3x20 mL). The organic phases were combined, dried over MgSO4,
filtered and concentrated in vacuo. Purification by flash column
chromatography afforded the corresponding β-keto dioxinone.
Chamber B: When the chamber A was sealed, THF (20 mL) was
added to a solid mixture of CO-gen.5 (0.64 g, 2.6 mmol, 1.3 equiv.)
and CsF (40 mg, 0.2 mmol, 0.2 equiv.) and the chamber was sealed.
General procedure C: Argon-flushed Suzuki-Miyaura cross-
coupling on brominated β-keto-dioxinones.
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10.1002/chem.201902731
Chemistry - A European Journal
FULL PAPER
To a solution of brominated β-keto dioxinone (1 equiv.) and boronic
acid (1.5 equiv.) in degassed THF/H2O (4:1, v/v, 0.2 M) were added
Pd(SPhos) (0.1 equiv.) and K3PO4 (1.5 equiv.). After 18 h at 40 °C the
reaction mixture was cooled down to RT and a 1M solution of HCl (20
mL) was added. The mixture was extracted with EtOAc (3x50 mL).
The organic phases were combined, dried over MgSO4, filtered and
concentrated in vacuo. Purification by flash column chromatography
afforded the corresponding β-keto dioxinone.
151.2, 150.1, 147.8, 147.6, 146.2, 141.9, 137.6, 134.6, 134.5, 129.5,
128.7, 128.0, 126.5, 124,7, 124.1, 123.9, 122.9, 121.6, 121.4, 120.0,
107.5, 97.2, 68.4, 58.5, 51.7, 43.6, 39.6, 25.2. IR (neat, cm-1): 2924,
2246, 1719, 1715, 1514, 1327, 1248, 1198, 1108, 1028. LRMS (ESI+,
acetonitrile): m/z (%) = 694 (20) [M+H+]. HRMS (ESI): m/z calcd. for
[C45H44NO6+] 694.3169, found 694.3160.
9,9-bis(2-methoxyethyl)-9H-fluorene (19) [204265-05-4]: To
a
solution of fluorene (13) (2.0 g, 12.0 mmol) in THF (30 mL) was added
at 0 °C t-BuOK (4.0 g, 36.0 mmol). After 10 min at 0 °C a solution of
2-methoxyethyl 4-methylbenzenesulfonate (5.5 g, 24.0 mmol) in THF
(10 mL) was added at 0 °C. After 1 h at 0 °C the reaction mixture was
warmed up to RT. After 1 h at RT a 1 M solution of HCl (40 mL) was
added and the mixture was extracted with EtOAc (3x 60 mL). The
organic phases were combined, dried over MgSO4, filtered and
concentrated in vacuo. Purification by flash column chromatography
(cyclohexane/EtOAc 90/10) afforded the product (2.0 g, 7.1 mmol,
60%) as a yellow oil. Data were in agreement with those reported in
literature.38
6-(2-(9,9-dibutyl-7-(4-methoxyphenyl)-9H-fluoren-2-yl)-2-
oxoethyl)-2,2-dimethyl-4H-1,3-dioxin-4-one
(4b):
Synthesized
according to General procedure B from dioxinone (15) and boronic
acid (16). Flash column chromatography: cyclohexane/EtOAc 90/10 to
70/30, yellow oil. Yield: 80%. 1H NMR (300 MHz, CDCl3): δ 7.94 (s,
1H), 7.91 (d, J = 1.6 Hz), 7.79 (t, J = 7.0 Hz, 2H), 7.64-7.59 (m, 2H),
7.56 (dd, J = 10.1, 1.4 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 5.47 (s, 1H),
3.98 (s, 2H), 3.88 (s, 3H), 2.04 (t, J = 8.2 Hz, 4H), 1.74 (s, 6H), 1.14-
1.01 (m, 4H), 0.66 (t, J = 7.3 Hz, 6H), 0.61-0.51 (m, 4H). 13C NMR (76
MHz, CDCl3): δ 193.1, 165.8, 161.1, 159.5, 152.7, 151.7, 147.0, 141.8,
138.1, 134.3, 133.7, 128.4, 128.3, 126.1, 122.5, 121.3, 119.7, 114.4,
107.4, 97.0, 55.5, 55.4, 43.6, 40.2, 26.1, 25.1, 23.1, 13.9. IR (neat,
cm-1): 1720, 1683, 1603, 1517, 1464, 1376, 1247, 1209, 1178, 1087,
1044. LRMS (ESI+, acetonitrile): m/z (%) = 575 (30) [M+Na+]. HRMS
(ESI): m/z calcd. for [C36H41O5+] 553.2954, found 553.2947.
2-iodo-9,9-bis(2-methoxyethyl)-9H-fluorene (20) [204265-20-3]: To
a solution of fluorene (19) (0.2 g, 0.71 mmol) and NIS (0.18 g, 0.78
mmol) in MeCN (10 mL) was added TFA (16 µL, 0.21 mmol). After 4 h
at 80 °C the reaction mixture was cooled down to RT and H2O (20
mL) was added. The mixture was extracted with EtOAc (3x50 mL).
The organic phases were combined, washed with a 10% solution of
Na2SO3, dried over MgSO4, filtered and concentrated in vacuo.
Purification by flash column chromatography (cyclohexane/EtOAc
90/10) afforded the product (0.17 g, 0.42 mmol, 59%) as a colourless
oil. 1H NMR (300 MHz, CDCl3): δ 7.76 (d, J = 1.3 Hz, 1H), 7.71-7.64
(m, 2H), 7.46-7.40 (m, 2H), 7.35 (dd, J = 5.5, 3.4 Hz, 2H), 3.01 (s, 6H),
2.71-2.63 (m, 4H), 2.33 (ddd, J = 9.2, 6.3, 2.0 Hz, 4H). 13C NMR (76
MHz, CDCl3): δ 151.4, 148.5, 140.2, 139.5, 136.5, 132.4, 128.2, 127.7,
123.1, 121.7, 120.1, 68.4, 58.5, 51.4, 39.6. IR (neat, cm-1): 1449,
1409, 1393, 1244, 1112, 1002. LRMS (ESI+, acetonitrile): m/z (%) =
431 (10) [M+Na+].
6-(2-(9,9-dibutyl-7-(4-(diphenylamino)phenyl)-9H-fluoren-2-yl)-2-
oxoethyl)-2,2-dimethyl-4H-1,3-dioxin-4-one
(4c):
Synthesized
according to General procedure C from dioxinone (15) and boronic
acid (17). Flash column chromatography: cyclohexane/EtOAc 90/10 to
70/30, yellow oil. Yield: 43%. 1H NMR (300 MHz, CDCl3): δ 7.94 (s,
1H), 7.91 (d, J = 1.7 Hz, 1H), 7.79 (t, J = 7.5 Hz, 2H), 7.63-7.52 (m,
3H), 7.33-7.28 (m, 2H), 7.20-7.13 (m, 5H), 7.05 (ddd, J = 8.4, 2.3,1.2
Hz, 2H), 5.47 (s, 1H), 3.98 (s, 2H), 2.15-1.93 (m, 4H), 1.73 (s, 6H),
1.14-1.01 (m, 4H), 0.66 (t, J = 7.3 Hz, 6H), 0.61-0.53 (m, 4H). 13C
NMR (76 MHz, CDCl3): δ 193.1, 165.7, 161.0, 152.9, 151.8, 147.7,
147.0, 141.5, 138.3, 134.9, 134.4, 129.5, 128.3, 128.0, 126.0, 124.7,
124.9, 123.3, 122.6, 121.4, 121.2, 119.8, 107.5, 97.1, 55.6, 43.7, 40.2,
27.1, 25.2, 23.0, 14.0. IR (neat, cm-1): 2931, 1731, 1676, 1593, 1514,
1492, 1468, 1331, 1280. LRMS (ESI+, acetonitrile): m/z (%) = 690
(50) [M+H+]. HRMS (ESI): m/z calcd. for [C47H47NO4+] 689.3505,
found 689.3515.
2-bromo-9,9-bis(2-methoxyethyl)-9H-fluorene (21) [649724-44-7]:
To a solution of fluorene (19) (1.5 g, 5.3 mmol) in DMF (50 mL) was
added NBS (1.0 g, 5.8 mmol). After 18 h at 60 °C NBS (0.5 g, 2.7
mmol) was added at 60 °C. After 2 h at 60°C the reaction mixture was
cooled down to RT and H2O (50 mL) was added. The mixture was
extracted with EtOAc (3x50 mL). The organic phases were combined,
washed with a 10% solution of Na2SO3 (2x30 mL), dried over MgSO4,
filtered and concentrated in vacuo. Purification by flash column
chromatography (cyclohexane/EtOAc 90/10) afforded the product
(0.97 g, 2.7 mmol, 50%) as a pale-yellow oil. 1H NMR (300 MHz,
CDCl3): δ 7.70 -7.65 (m, 1H), 7.56 (dd, J = 4.9, 2.8 Hz, 2H), 7.48 (dd,
J = 8.1, 1.7 Hz, 1H), 7.44-7.39 (m, 1H), 7.39-7.31 (m, 2H), 3.02 (s,
6H), 2.71-2.62 (m, 4H), 239-2.26 (m, 4H). 13C NMR (76 MHz, CDCl3):
δ 151.3, 148.7, 139.6, 139.5, 130.7, 128.0, 127.7, 126.6, 123.2, 121.4,
121.3, 120.1, 68.4, 58.5, 51.5, 39.7. IR (neat, cm-1): 1727, 1607, 1645,
1375, 1276, 1202, 1113, 1017. LRMS (ESI+, acetonitrile): m/z (%) =
383 (20%) [M+Na+].
6-(2-(9,9-bis(2-methoxyethyl)-7-(4-methoxyphenyl)-9H-fluoren-2-
yl)-2-oxoethyl)-2,2-dimethyl-4H-1,3-dioxin-4-one (5b): Synthesized
according to General procedure C from dioxinone (23) and boronic
acid (16). Flash column chromatography: cyclohexane/EtOAc 70/30 to
50/50, yellow oil. Yield: 62%. 1H NMR (300 MHz, CDCl3): δ 8.03 (d, J
= 1.1 Hz, 1H), 7.96 (dd, J = 8.0, 1.6 Hz, 1H), 7.84-7.77 (m, 2H), 7.62
(dd, J = 8.3,1.6 Hz, 4H), 7.02 (d, J = 8.8 Hz, 2H), 5.46 (s, 1H), 3.97 (s,
2H), 3.88 (s, 3H), 2.99 (s, 6H), 2.79-2.62 (m, 4H), 2.43 (t, J = 7.6 Hz,
4H), 1.75 (s, 6H). 13C NMR (76 MHz, CDCl3): δ 192.8, 165.5, 159.6,
151.4, 146.3, 142.1, 137.5, 134.7, 133.3, 128.7, 128.4, 126.6, 122.9,
121.6, 121.5, 120.0, 114.6, 107.5, 97.2, 68.5, 58.6, 55.4, 51.7, 43.7,
39.4, 25.0. IR (neat, cm-1): 1726, 1682, 1636, 1603, 1517, 1464, 1407,
1390, 1247, 1177, 1024. LRMS (ESI+, acetonitrile): m/z (%) = 557
(20) [M+H+]. HRMS (ESI): m/z calcd. for [C34H37O7+] 557.2539, found
557.2527.
2-bromo-7-iodo-9,9-bis(2-methoxyethyl)-9H-fluorene (22): To
a
solution of fluorene (21) (1.0 g, 2.77 mmol) in AcOH/H2O/H2SO4
(10:5:1 v/v, 24.6 mL) were added I2 (0.34 g, 1.35 mmol) and KIO3
(144 mg, 0.68 mmol). After 2 h at 90 °C the reaction mixture was
cooled down to RT and H2O (30 mL) was added. The mixture was
extracted with EtOAc (3x60 mL). The organic phases were combined,
washed with a 10% solution of Na2SO3 (2x50 mL), dried over MgSO4,
filtered and concentrated in vacuo. Purification by flash column
chromatography (cyclohexane/EtOAc 90/10) afforded the product (1.0
g, 2.05 mmol, 74%) as a colourless oil. 1H NMR (300 MHz, CDCl3):
δ .75 (d, J = 1.1 Hz, 1H), 7.71-7.66 (m, 1H), 7.57-7.51 (m, 2H), 7.48
(dd, J = 8.1, 1.6 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 3.03 (s, 6H), 2.82-
2.59 (m, 4H), 2.41-2.22 (m, 4H). 13C NMR (76 MHz, CDCl3): δ 151.0,
6-(2-(7-(4-(diphenylamino)phenyl)-9,9-bis(2-methoxyethyl)-9H-
fluoren-2-yl)-2-oxoethyl)-2,2-dimethyl-4H-1,3-dioxin-4-one
Synthesized according to General procedure C from dioxinone (23)
and boronic acid (17). Flash column chromatography:
(5c):
cyclohexane/EtOAc 70/30, yellow oil. Yield: 41%. 1H NMR (300 MHz,
CDCl3): δ 8.03 (s, 1H), 7.96 (dd, J = 7.8, 1.6 Hz, 1H), 7.80 (dd, J = 8.2,
6.0 Hz, 2H), 7.66 (s, 2H), 7.63-7.60 (m, 1H), 7.56 (d, J = 8.6 Hz, 2H),
7.30 (d, J = 8.4 Hz, 2H), 7.20-7.12 (m, 6H), 7.09-7.03 (m, 3H), 5.46 (s,
1H), 3.97 (s, 2H), 2.99 (s, 6H), 2.81-2.61 (m, 4H), 2.42 (t, J = 7.2 Hz,
4H), 1.75 (s, 6H). 13C NMR (76 MHz, CDCl3): δ 192.8, 165.5, 160.9,
This article is protected by copyright. All rights reserved.

10.1002/chem.201902731
Chemistry - A European Journal
FULL PAPER
150.7, 139.1, 138.5, 136.7, 132.5, 130.8, 126.5, 122.0, 121.7, 121.4,
93.3, 68.2, 58.5, 51.7, 39.4. HRMS (ESI): m/z calcd. for
[C19H21O2(79)Br+] 488.9749, found 488.9798.
27.1, 26.6, 26.3, 26.1, 23.1, 13.9. IR (neat, cm-1): 3035, 2955, 2929,
2859, 1758, 1712, 1587, 1514, 1491, 1463, 1246, 1178, 1091, 1027.
LRMS (ESI+, acetonitrile): m/z (%) = 837 (100) [M+H+]. HRMS (ESI):
m/z calcd. for [C56H54NO6+] 836.3951, found 836.3970.
General procedure D: Condensation between β-keto-dioxinones
and keto-alcohol (3).
(Z)-3-(3-(9,9-bis(2-methoxyethyl)-9H-fluoren-2-yl)-1-hydroxy-3-
oxoprop-1-en-1-yl)-6,6,9a-trimethyl-5,6-dihydro-2H-furo[3,2-
g]isochromene-2,9(9aH)-dione (25a): Synthesized according to
To a solution of dioxinone (1.2 equiv.) and alcohol (3) (1 equiv.) in
toluene (0.1 M) was added 4Å molecular sieves (100 mg per mmol).
After 30 min at 110 °C Et3N (2.5 equiv.) was added at 110 °C. After 30
min at 110 °C the reaction mixture was cooled down to RT, a 1 M
solution of HCl was added and the mixture was vigorously stirred for
15 min. The mixture was extracted with EtOAc (3x20 mL), the organic
phases were combined, dried over MgSO4, filtered and concentrated
in vacuo. Purification by flash column chromatography afforded the
corresponding analogue.
General procedure
D
from dioxinone (5a). Flash column
chromatography: cyclohexane/EtOAc 80/20 to 70/30, orange oil.
Yield: 9%. 1H NMR (300 MHz, CDCl3): δ 8.10 (d, J = 1.0 Hz, 1H),
8.04 (dd, J = 8.1, 1.5 Hz, 1H), 7.81-7.75 (m, 3H), 7.56 (s, 1H), 7.51-
7.36 (m, 4H), 7.14 (d, J = 1.3 Hz, 1H), 2.99 (s, 6H), 2.80 (d, J = 16.6
Hz, 1H), 2.76-2.62 (m, 6H), 2.51-2.30 (m, 4H), 1.74 (s, 3H), 1.47 (s,
3H), 1.42 (s, 3H). 13C NMR (76 MHz, CDCl3): δ 190.4, 189.9, 174.1,
168.4, 167.9, 158.9, 150.5, 149.6, 145.6, 141.2, 139.3, 134.9, 129.0,
128.1, 127.8, 123.4, 122.4, 121.1, 120.0, 114.8, 113.6, 110.8, 97.5,
86.7, 81.4, 68.5, 58.5, 51.6, 40.1, 39.5, 28.1, 26.6, 26.3. IR (neat, cm-
1): 2929, 1755, 1711, 1587, 1449, 1354, 1266, 1178, 1113, 1092.
LRMS (ESI+, acetonitrile): m/z (%) = 597 (100) [M+H+]. HRMS (ESI):
m/z calcd. for [C36H37O8+] 597.2488, found 597.2499.
(Z)-3-(3-(9,9-dibutyl-9H-fluoren-2-yl)-1-hydroxy-3-oxoprop-1-en-1-
yl)-6,6,9a-trimethyl-5,6-dihydro-2H-furo[3,2-g]isochromene-
2,9(9aH)-dione (24a): Synthesized according to General procedure
D
from dioxinone (4a). Flash column chromatography:
cyclohexane/EtOAc 90/10 to 70/30, brown oil. Yield: 8%. 1H NMR
(300 MHz, CDCl3): δ 8.03 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 (s, 1H), 7.77
(dd, J = 4.5, 3.3 Hz, 3H), 7.57 (s, 1H), 7.39-7.35 (m, 3H), 7.13 (d, J =
1.4 Hz, 1H), 2.79 (d, J = 17.2 Hz, 1H), 2.72 (d, J = 17.0 Hz, 1H), 2.10-
1.96 (m, 4H), 1.74 (s, 3H), 1.47 (s, 3H), 1.42 (s, 3H), 1.10-1.03 (m,
(Z)-3-(3-(9,9-bis(2-methoxyethyl)-7-(4-methoxyphenyl)-9H-fluoren-
2-yl)-1-hydroxy-3-oxoprop-1-en-1-yl)-6,6,9a-trimethyl-5,6-dihydro-
2H-furo[3,2-g]isochromene-2,9(9aH)-dione (25b): Synthesized
according to General procedure D from dioxinone (5b). Flash column
chromatography: cyclohexane/EtOAc 80/20 to 70/30, orange oil.
Yield: 4%. 1H NMR (300 MHz, CDCl3): δ 8.11 (d, J = 1.1 Hz, 1H),
8.05 (dd, J = 8.1, 1.5 Hz, 1H), 7.97-7.91 (m, 1H), 7.82-7.74 (m, 2H),
7.65-7.58 (m, 4H), 7.57 (s, 1H), 7.14 (s, 1H), 7.02 (d, J = 1.3 Hz, 2H),
3.88 (s, 3H), 3.01 (s, 6H), 2.77-2.71 (m, 6H), 2.46-2.40 (m, 4H), 1.75
(s, 3H), 1.48 (s, 3H), 1.41 (s, 3H). 13C NMR (76 MHz, CDCl3): δ 190.3,
189.9, 174.1, 167.9, 159.6, 158.9, 151.2, 149.9, 145.4, 141.7, 141.2,
137.9, 134.7, 133.6, 128.4, 128.2, 122.3, 121.5, 121.3, 120.0, 114.8,
114.5, 113.6, 110.8, 97.5, 86.6, 81.3, 68.5, 58.5, 55.6, 51.6, 40.2,
39.6, 28.1, 26.6, 26.3. IR (neat, cm-1): 2928, 1727, 1601, 1558, 1464,
1452, 1272, 1203, 1121, 1073. LRMS (ESI+, acetonitrile): m/z (%) =
703 (100) [M+H+]. HRMS (ESI): m/z calcd. for [C43H43O9+] 703.2907,
found 703.2922.
4H), 0.88-0.83 (m, 4H), 0.65 (t, J = 7.3 Hz, 6H), 0.67-0.56 (m, 4H). 13
C
NMR (76 MHz, CDCl3): δ 190.9, 189.9, 173.7, 168.5, 167.7, 158.8,
152.2, 151.4, 146.5, 141.1, 140.0, 134.6, 128.6, 127.7, 127.2, 123.2,
122.0, 120.9, 119.8, 114.9, 113.6, 110.8, 97.6, 86.6, 81.3, 55.4, 40.1,
28.1, 26.6, 26.3, 26.1, 23.1, 13.9. IR (neat, cm-1): 2956, 2930, 2859,
1758, 1712, 1587, 1466, 1373, 1353, 1265, 1250, 1178, 1091, 1023.
LRMS (ESI+, acetonitrile): m/z (%) = 615 (100) [M+Na+]. HRMS (ESI):
m/z calcd. for [C38H41O6+] 593.2903, found 593.2916.
(Z)-3-(3-(9,9-dibutyl-7-(4-methoxyphenyl)-9H-fluoren-2-yl)-1-
hydroxy-3-oxoprop-1-en-1-yl)-6,6,9a-trimethyl-5,6-dihydro-2H-
furo[3,2-g]isochromene-2,9(9aH)-dione
(24b):
Synthesized
according to General procedure D from dioxinone (4b). Flash column
chromatography: cyclohexane/EtOAc 95/5 to 85/15, orange oil. Yield:
11%. 1H NMR (300 MHz, CDCl3): δ 8.04 (dd, J = 8.1, 1.5 Hz, 1H),
8.00 (d, J = 1.0 Hz, 1H), 7.83-7.75 (m, 3H), 7.64 (m, 6H), 7.14 (d, J =
1.3 Hz, 1H), 7.06-6.99 (m, 2H), 3.88 (s, 3H), 2.79 (d, J = 17.1 Hz, 1H),
2.72 (d, J = 17.3 Hz, 1H), 2.11-1.98 (m, 4H), 1.75 (s, 3H), 1.47 (s, 3H),
1.41 (s, 3H), 1.08 (dq, J = 14.6, 7.2 Hz, 4H), 0.93-0.79 (m, 4H), 0.66 (t,
J = 7.3 Hz, 6H), 0.62-0.55 (m, 4H). 13C NMR (76 MHz, CDCl3): δ
190.9, 189.9, 173.7, 168.5, 167.7, 159.5, 158.9, 152.9, 151.5, 146.3,
141.3, 141.1, 138.7, 134.4, 134.0, 128.4, 127.8, 126.0, 122.0, 121.3,
121.2, 119.8, 114.9, 114.4, 113.6, 110.8, 97.6, 86.6, 81.3, 55.6, 40.2,
28.1, 27.1, 26.7, 26.3, 26.1, 23.1, 13.9. IR (neat, cm-1): 2930, 1758,
1713, 1589, 1518, 1464, 1355, 1249, 1178, 1091, 1026. LRMS (ESI+,
acetonitrile): m/z (%) = 699 (100) [M+H+]. HRMS (ESI): m/z calcd. for
[C45H47O7+] 699.3322, found 699.3325.
(Z)-3-(3-(7-(4-(diphenylamino)phenyl)-9,9-bis(2-methoxyethyl)-9H-
fluoren-2-yl)-1-hydroxy-3-oxoprop-1-en-1-yl)-6,6,9a-trimethyl-5,6-
dihydro-2H-furo[3,2-g]isochromene-2,9(9aH)-dione
(25c):
Synthesized according to General procedure D from dioxinone (5c).
Flash column chromatography: cyclohexane/EtOAc 80/20 to 70/30,
orange oil. Yield: 5%. 1H NMR (300 MHz, CDCl3): δ 8.11 (d, J = 1.0
Hz, 1H), 8.05 (dd, J = 8.1, 1.5 Hz, 1H), 7.82-7.76 (m, 3H), 7.66-7.61
(m, 2H), 7.58 -7.55 (m, 3H), 7.31-7.29 (m, 4H), 7.19-7.14 (m, 7H),
7.08-7.03 (m, 2H), 3.01 (s, 6H), 2.81-2.67 (m, 6H), 2.52-2.34 (m, 4H),
1.75 (s, 3H), 1.48 (s, 3H), 1.43 (s, 3H). 13C NMR (76 MHz, CDCl3): δ
192.3, 189.9, 174.1, 168.4, 167.9, 158.9, 151.2, 149.9, 147.7, 145.4,
141.5, 141.2, 138.1, 134.8, 134.7, 129.5, 128.2, 128.0, 126.4, 124.7,
124.0, 123.2, 122.3, 121.4, 120.0, 114.8, 113.6, 110.8, 97.5, 86.6,
81.4, 68.5, 58.5, 51.6, 40.2, 39.6, 28.1, 27.1, 26.6, 26.3. IR (neat, cm-
1): 2932, 1758, 1706, 1590, 1519, 1492, 1269, 1179, 1112, 1109.
LRMS (ESI+, acetonitrile): m/z (%) = 840 (100) [M+H+]. HRMS (ESI):
m/z calcd. for [C54H50NO8+] 840.3536, found 840.3530.
(Z)-3-(3-(9,9-dibutyl-7-(4-(diphenylamino)phenyl)-9H-fluoren-2-yl)-
1-hydroxy-3-oxoprop-1-en-1-yl)-6,6,9a-trimethyl-5,6-dihydro-2H-
furo[3,2-g]isochromene-2,9(9aH)-dione
(24c):
Synthesized
according to General procedure D from dioxinone (4c). Flash column
chromatography: cyclohexane/EtOAc 90/10 to 70/30, orange oil.
Yield: 10%. 1H NMR (300 MHz, CDCl3): δ 8.05 (dd, J = 8.1, 1.4 Hz,
1H), 8.01 (s, 1H), 7.83-7.74 (m, 3H), 7.63-7.53 (m, 6H), 7.32-7.26 (m,
5H), 7.20-7.12 (m, 8H), 7.04 (dd, J = 10.3, 4.2 Hz, 3H), 2.80 (d, J =
17.2 Hz, 1H), 2.73 (d, J = 17.1 Hz), 2.11-1.99 (m, 4H), 1.75 (s, 3H),
1.48 (s, 3H), 1.41 (s, 3H), 1.10-1.05 (m, 4H), 0.67 (t, J = 7.3 Hz, 6H),
0.64-0.55 (m, 4H). 13C NMR (76 MHz, CDCl3): δ 190.9, 189.9, 173.7,
168.5, 167.7, 158.8, 152.9, 151.6, 147.8, 147.5, 141.1, 138.8, 135.2,
134.4, 129.5, 128.0, 127.8, 125.9, 124.6, 124.0, 123.2, 122.0, 121.2,
121.1, 119.8, 114.9, 113.6, 110.8, 86.6, 81.3, 55.5, 40.2, 40.1, 28.1,
1P-spectroscopy
UV-visible absorption spectra were obtained on a Agilent Cary 60
scan spectrophotometer by using a rectangular quartz cell (light path
10 × 10 mm, chamber volume: 3.5 mL). O.D. was comprised between
1.5 and 0.7. Fluorescence measurements were conducted on a
HORIBA Fluorolog
3 spectrophotometer applying inner spectral
correction factor and with a quartz fluorescence cell (Hellma, 104F-
QS, light path: 10 × 10 mm, chamber volume: 3500 µL, excitation and
emission slit: 2 nm). Fluorescence quantum yields were measured by
This article is protected by copyright. All rights reserved.

10.1002/chem.201902731
Chemistry - A European Journal
FULL PAPER
linear regression of the integral of the fluorescence signal plotted
against absorption factor (Fabs=1-10-Abs) keeping Abs<0.1.
laser (Coherent Chameleon Ultra II, 80 MHz, 140 fs) in the range
720–990 nm. The excitation beam (3 mm diameter) was focused with
a lens (focal length 10 cm) in fluorescence cells (10×10 mm). The
fluorescence was collected at right-angle by a ×10 Zeiss microscope
objective coupled to a fiber optic spectrometer (Ocean Optics S2000).
After verifying that the fluorescence intensity exhibited quadratic
intensity dependence for each sample, the incident beam intensity
was adjusted to 40 mW. To avoid thermal effects, a magnetic stirrer
continuously stirred the solutions. Calibration of the spectra was
performed by comparison with the two-photon absorption Spectra of
Styryl 9M,32 Coumarin 510, and 540 (common references in our
laboratory, not published but measured and calibrated thanks to the
published spectra of Coumarin 307 and fluorescein).31
Cell Culture
As previously described, PC12 cells were plated on glass bottom
dishes (MatTek Corporation, MA, USA) for two-photon microscopy
(SP8) or on 24-well plates (Costar, Illkirch, France) for wide-field
microscopy (CD7) and cultured in a humidified incubator at 37°C in an
atmosphere of 5% CO2 39. Cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM) (Thermo Fisher Scientific, Illkirch, France)
supplemented with 7% heat-inactivated fetal bovine serum (Sigma-
Aldrich), 7% horse serum (Lonza Bioscience, Walkersville, MD, USA),
2.5% HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)
(Thermo Fisher Scientific), 1% glutamine (Thermo Fisher Scientific),
100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher
Scientific).
Cytotoxicity Assay
PC12 cells were prepared for cell viability studies in 24-well plates.
The cells were incubated for 1 or 24 hours with a single epicocconone
analogue (2, 25a, 25b or 25c) at a concentration of 10-6 M. Two drops
of NucGreenTM Dead 488 ReadyProbesTM reagent (Thermo Fisher
Scientific) per ml of media were added into wells, followed by further
incubation for 10 min at 37°C. Since the dye is cell-impermeant and
emits green fluorescence when bound to DNA, the viability was
determined by imaging through transmitted and fluorescence modes,
positive cells on an automated boxed inverted Zeiss CD7 microscope
piloted with Zen software (Roper Scientific, Evry, France). Statistical
Two-photon Microscopy
2-photon (2P) microscopy was performed on a fixed stage Leica TCS
SP8 confocal microscope (Leica Microsystems, Nanterre, France)
equipped with a femtosecond laser source (Chameleon Vision II,
Coherent, Les Ulis, France, 80 MHz, 140 fs) and conventionnal
scanner (400 Hz). Using a 25x objective (0.95, water immersion),
fluorescence emission was detected through a hybrid detector (HyD)
in photon counting mode. Excitation wavelength (775 nm) and
emission detection (570-640 nm) were similar to those previously
determined for analogue 2.8
analysis was performed with the GraphPad Prism
4 software
(GraphPad Software Inc.) and one-way Analysis of Variance
a
(ANOVA) with a Tukey-Kramer multiple comparisons tests was used.
Prior dilution in culture medium, solubilization of dyes was facilitated
with the non-ionic surfactant polyol Pluronic® F-127 (ThermoFisher
Scientific, Montigny le Bretonneux, France) (v/v) (Fig. S6 for 25a).
Measurements were carried out in phenol red-free cell culture medium
(final dye concentration: 10-6 M) or in labelled cells (final dye
concentration: 10-6 M). For live cell imaging, cells were incubated with
dyes during 45 min was performed in phenol red-free Dulbecco’s
modified Eagle’s medium (DMEM) rinsed once and observed. Large
view (Image Size = 591.05 x 591.05 µm at 1024 x 1024 pixels; optical
zoom = 0.75), higher magnification (Image Size = 73.88 x 73.88 µm at
512 x 512 pixels; optical zoom = 3.0) and ZX/ZY projections were
used to illustrate cell population labelling and subcellular details.
Acknowledgements
This work was partially supported by Normandie Université
(NU), the Région Normandie, the Centre National de la
Recherche Scientifique (CNRS), Université de Rouen
Normandie (URN), INSA Rouen Normandie, Innovation
Chimie Carnot (I2C) and Labex SynOrg (ANR-11-LABX-
0029) for financial support to R.M. We are grateful to Patricia
Martel (University of Rouen Normandy) for IR analyses, and
to Albert Marcual (CNRS) for HRMS analyses.
Image Analysis
Keywords: epicocconone • two-photon • cell imaging •
Images of epicocconone analogues-labeled PC12 cells were obtained
with strictly identical acquisition parameters including objective, laser
power and emission band. In addition, fluorescence collection was
performed using a very sensitive hybrid detector in photon counting
mode. Therefore, fluorescence intensities of epicocconone analogues
(2, 25a, 25b and 25c) in cellulo were extracted from full images
(n=12), determined by the number of photons received by the detector
and converted in a gray value. Fluorescence intensity obtained with
analogue 2 was considered as a reference i.e. 100% and other
intensities were normalized and compared to analogue 2. Statistical
analysis by using t-test with the Kolmogorov and Smirnov method was
performed with the GraphPad Prism 4 software (GraphPad Software
Inc., San Diego, CA, USA). ImageJ (Rasband, W.S., U. S. National
Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/,
1997-2016) was used to adjust image brightness and contrast and to
perform z projections of 3D images (xyz).
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The modification of the skeleton of the
Samuel Oger, Damien Schapman,
natural product epicocconone by
Romain Mougeot, Stéphane Leleu,
Noëlle Lascoux, Patrice Baldeck,
Magalie Bénard, Thibault Gallavardin,
Ludovic Galas, Xavier Franck*
O
O
replacing the triene side chain by a
fluorenyl scaffold can noticeably
increase the fluorophore’s absorption
coefficient. Two-photon absorption
cross section measurements of the
fluorenyl epicocconone analogues
O
O
O
OH
OH
epicocconone
O
Page No. – Page No.
O
O
O
R1
R1
Two-photon absorption and cell
imaging of fluorene-functionalized
epicocconone analogues
OH
O
show
a structure-dependency with
R2
fluorenyl synthetic analogues
designed for two-photon absorption and imaging
values ranging from 60 GM to 280 GM
and live cell imaging show intense
staining of intracellular vesicles-like
structures around the nucleus.
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Title
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