Cellular imaging using BODIPY-, pyrene- and phthalocyanine-based conjugates

Article abstract of DOI:10.1016/j.bmc.2017.11.050

Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in the green/red have been synthesized (as the form of dyads-pentads), studied by spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanin

Full text of DOI:10.1016/j.bmc.2017.11.050

Accepted Manuscript
Cellular imaging using BODIPY-, pyrene- and phthalocyanine- based conju-
gates
Faustine Bizet, Martin Ipuy, Yann Bernhard, Vivian Lioret, Pascale Winckler,
Christine Goze, Jean-Marie Perrier-Cornet, Richard A. Decréau
PII:
S0968-0896(17)31614-0
https://doi.org/10.1016/j.bmc.2017.11.050
BMC 14103
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
9 August 2017
13 November 2017
30 November 2017
Please cite this article as: Bizet, F., Ipuy, M., Bernhard, Y., Lioret, V., Winckler, P., Goze, C., Perrier-Cornet, J-M.,
Decréau, R.A., Cellular imaging using BODIPY-, pyrene- and phthalocyanine- based conjugates, Bioorganic &
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.050
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Graphical Abstract
Cellular imaging using BODIPY-, pyrene-
and phthalocyanine- based conjugates
Leave this area blank for abstract info.
Faustine Bizet, Martin Ipuy, Yann Bernhard, Vivian
Lioret, Pascale Winckler, Christine Goze, Jean-
Marie Perrier-Cornet, Richard A. Decréau
aICMUB Institute, University Bourgogne – Franche Comté, 9 Avenue Alain Savary, Sciences Mirande,
21078 Dijon (France) ; b AgroSup Dijon, University Bourgogne – Franche Comté, Dimacell Imaging
Ressource Center, UMR A 02.102 PAM, F-21000 Dijon (France)
F
N
B
F
N

Bioorganic & Medicinal Chemistry
jo u r n a l h o m e p a g e : w w w . e ls e v i e r . c o m
Cellular imaging using BODIPY-, pyrene- and phthalocyanine- based conjugates
Faustine Bizet,1 Martin Ipuy,1 Yann Bernhard,1 Vivian Lioret,1 Pascale Winckler,2 Christine Goze,1 Jean-
Marie Perrier-Cornet,2 Richard A. Decréau1*
a ICMUB Institute, University Bourgogne – Franche Comté, 9 Avenue Alain Savary, Sciences Mirande, 21078 Dijon (France)
b AgroSup Dijon, University Bourgogne – Franche Comté, Dimacell Imaging Ressource Center, UMR A 02.102 PAM, F-21000 Dijon (France)
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Fluorescent Probes aimed at absorbing in the blue/green region of the spectrum and emitting in
the green/red have been synthesized (as the form of dyads-pentads), studied by
spectrofluorimetry, and used for cellular imaging. The synthesis of phthalocyanine-pyrene 1
was achieved by cyclotetramerization of pyrenyldicyanobenzene, whereas phthalocyanine-
BODIPY 2c was synthesized by Sonogashira coupling between tetraiodophthalocyanine and
meso-alkynylBODIPY. The standard four-steps BODIPY synthesis was applied to the BODIPY-
Available online
1
pyrene dyad 3 starting from pyrenecarbaldehyde and dimethylpyrrole. H, 13C, 19F, 11B-NMR,
ICP, MS, and UV/Vis spectroscopic analyses demonstrated that 2c is a mixture of BODIPY-Pc
conjugates corresponding to an average ratio of 2.5 BODIPY per Pc unit, where its bis, tris,
tetrakis components could not be separated. Fluorescence emission studies (M concentration
in THF) showed that the design of the probes allowed excitation of their antenna (pyrene,
BODIPY) in the blue/green region of the spectrum, and subsequent transfer to the acceptor
platform (BODIPY, phthalocyanine) followed by its emission in the green/red (with up to 140-
350 nm overall Stokes shifts). The fluorescent probes were used for cellular imaging of B16F10
melanoma cells upon solubilization in 1% DMSO containing RPMI or upon encapsulation in
liposomes (injection method). Probes were used at 1-10 M concentrations, cells were fixed
with methanol and imaged by biphoton and/or confocal microscopy, showing that probes could
achieve the staining of cells membranes and not the nucleus.
Keywords:
Dyad/pentad syntheses
Energy transfer
Phthalocyanine-BODIPY
BODIPY-Pyrene
Phthalocyanine-Pyrene
Spectrofluorimetry
Fuorescence cellular imaging
2009 Elsevier Ltd. All rights reserved.
1. Introduction
between the BODIPY and the alkyne, whereas our system 2c
provides a shorter connection between Pc and BODIPY that are
In biphoton imaging, deeper penetration of light in tissues is
possible compared to monophoton excitation [1]. Conjugating a
blue-absorbing dye to another NIR-emitting-dye will make
emission in the NIR possible provided that either FRET or
through bond energy transfers (TBET) occur. Hence, such a
strategy is a two-fold approach that will minimize the
autofluorescence on the way in (biphoton) and way out (TBET
followed by emission in the NIR). The rational of our study was
to develop dyads/pentads of fluorophores in order to harvest light
on a large window in biphoton microscopy. We selected three
well-known classes of fluorophores that span the optical window:
Pyrene (380 nm, blue), BODIPY (500 nm, green), and
Phthalocyanine (Pc, 680 nm, near-IR) [2] (which are also known
as robust and efficient photosensitizers in photodynamic therapy
[3ab]). Hence, we were interested in fluorophore conjugates that
could be used in cellular imaging upon transferring light from
300 nm to 700 nm (as in Pyrene-Pc 1a reported by Ozcesmeci et
al. [4]), 500 nm to 700 nm (as in BODIPY-Pc 2c) and 300 nm to
500 nm (as in Pyrene-BODIPY 3a reported by Goze et al. [5]).
Some of these fluorescent molecular assemblies have been
previously reported but have not been used as probes for cellular
imaging. However, the design of BODIPY-Pc conjugates 2a and
2b reported by Göl et al. [6a] and Osati et al. [6b], respectively,
are slightly different than 2c depicted in Figure 1. Göl et al.
BODIPY-Pc conjugate 2b has either one phenyl ring or a -CH2O-
separated with only one alkynyl group. The rationale for such an
optimized design was to hope for a better transfer between the
two antennas within the dyad by increasing the conjugation
between the two antennas. At this stage of the study the yields of
transfers were not measured. [7]
2. Materials and Methods
2.1. Materials: Chemicals were used as provided.
2.2. Measurements and Instrumentations
LC-MS mass spectrometry analyses, Analytical and reverse
phase HPLC were performed on a Ultimate 3000 Thermo
DIONEX apparatus. MALDI-TOF mass spectrometry analyses
were performed on an Ultraflex II LRF 2000 apparatus, using
dithranol and DHB as a matrix. ICP analyses were performed on
a ICP-AES iCAP 7400 double visée Thermo apparatus. Low-
resolution ESI analyses were carried out on an Amazon SL
(BRUKER) mass spectrometer. High-resolution ESI analyses
were performed on a LTQ Orbitrap XL (THERMO) apparatus.
1H RMN , 19F NMR and 11B NMR analyses were performed on a
300 MHz Bruker Avance III NanoBay NMR apparatus. UV/Vis
absorption spectra were measured using a UV-Visible AGILENT
CARY 50 spectrophotometer. Fluorescence measurements,

Fig.1. Synthetic dyads/triads/pentads described in the literature (1, 2a, 3) and reported in this work (2c), that span the 300-700 nm spectrum region.
added to quench the reaction, the organic phase was extracted
with dichloromethane, washed with water and with a saturated
solution of ammonium chloride, and the solvents were removed
under reduced pressure. The resulting powder was washed with
methanol and dichloromethane. Yield = 60%
excitation and emission spectra were recorded on a Fluorolog
Jobin Yvon Horiba Xenon Lamp spectrometer. Biphotonic
images were collected on a Nikon A1-MP scanning microscope
(Nikon, Japan). Imaging was carried out with a ×25 Apo
LWD objective (NA: 1.1, Water Immersion, Nikon, Japan) at a
scanning speed of 0.5 frame per second. An IR laser
(Chameleon, Coherent) was used to provide a 780nm excitation.
Fluorescence emission was collected on four detection channels
(channel 1 (blue) : FF01-492/SP-25 filter [400-492 nm] ; channel
2 (green) : FF03-525/50-25 filter [500-550 nm] ; channel 3
(orange) : FF01-575/25-25 filter [563-588 nm] ; channel 4 (red)
FF01-629/56-25 filter [601-657 nm], Semrock). Confocal images
were collected using a Nikon C1Si Eclipse TE 2000 confocal
microscope (Nikon, Japan). Imaging was carried out with a ×100
PlanApo objective (NA: 1.4, oil, Nikon, Japan). Two laser
diodes were used as light sources, which delivered 488 and 561
nm wavelength light. Fluorescence emission was collected by a
spectral detector, using collection bands of [489-648]nm with
488nm excitation and [566-721]nm for 561nm excitation.
1H NMR (300 MHz, DMSO-d6, 300 K): δ (ppm)= 2.5 - 3 (broad
s, H from BODIPY methyl) ; 6.04 (broad s, H from BODIPY
core) (hard to define the exact number of proton, no well-define
peak), 7.0 – 8.5 (broad signal, H from Pc) 19F NMR (280 MHz,
d-DMSO, 300 K): δ (ppm)= -146 ppm (not well-defined
peak).11B NMR (128 MHz, d-DMSO, 300 K): δ (ppm)= 0.524
(not well-defined peak). MALDI-TOF: m/z= 1224.85
[Mmono+H]+; 1214.82 [Mmono-9]+, 1369.06 [Mbis+H]+; 1349.06
[M-F]+; 1359.03 [Mbis-9]+; 1513.26 [Mtris-9]+; 1493.26 [M-F]+;
1503.23
C92H68B4F8N16Zn: 1656.53 (exact mass); Mono 1223.88 ; Bis
1368.10 ; Tris 1512.32 ; Tetra 1656.23). ICP-AES: Zinc
[Mtris-9]+;
1647.42
[Mtetra-9]+(calcd
for
=
theorical
3.94%, Zinc exp= 3.83%, Boron theorical = 2.61%, Boron exp= 1.63%.
UV-Vis (DCM) : λmax (nm)= 374, 567, 697.
2.3. Methods
Pyrenyl-BODIPY(3):
2.3.1. Syntheses of compounds 1-9
Pyrenecarboxaldehyde (230 mg, 1 mmol) and dimethylpyrrole
(0.215 mL, 2 mmol) were put in a round bottom flask then 30 mL
of dry dichloromethane was added under nitrogen atmosphere,
followed by one drop of trifluoroacetic acid. The solution became
red, and the mixture was stirred for 16h under nitrogen
atmosphere. p-Chloranil (250 mg, 1 mmol) was added and the
solution was stirred for 30 min under nitrogen atmosphere.
Triethylamine (3 mL, 22 mmol) and boron trifluoride
diethyletherate (3 mL, 24 mmol) were added in the mixture that
was previously cooled in an ice bath. The mixture was stirred for
3 h at room temperature. Then the organic phase was washed
with water and dried with magnesium sulphate, and solvents
were removed under reduced pressure. The resulting crude
product was filtered on a plug of silica gel (dichloromethane).
The filtrates were collected, the solvent was removed, and the
resulting solid was subjected to silica gel column
chromatography (silica gel, eluent ethyl acetate : petroleum ether,
gradient from 6 :94 to 12 : 88 vol.). Yield = 22%.
[2,9(10),16(17),23(24)-tetramethoxypyrenylphthalocyanina
to]zinc(II) (1):
4-(Pyren-1-ylmethoxy)-phthalonitrile (5) (70 mg, 0.19 mmol),
zinc acetate (10 mg, 0.048 mmol), 1,8-diazabicyclo{5.4.0}undec-
7-ene (21 mg, 0.19 mmol) and pentanol (10 mL) were placed in a
round bottom flask. The mixture was heated to reflux at 140°C
for 22 h. The reaction mixture was subsequently allowed to come
to room temperature, and then an ice bath was added to
precipitate the product down. The precipitate was filtrated on a
Büchner, rinsed with water, methanol, ethanol and acetone. Yield
= 17%.
1H NMR (300 MHz, DMSO-d6, 300 K): δ (ppm)= 6.2 (s, 8H);
7.8 – 8.7 (m, 48H). MS (MALDI-TOF): m/z= 1497.89 [M+H]+,
·
·
1283.83 [M-(CH2-pyr)+H] , 1069.78 [M-(CH2-pyr)2+2H] ,
·
·
855.53 [M-(CH2-pyr)3+3H] , 641.13 [M-(CH2-pyr)4+4H] . (Calcd
for C100H57N8O4Zn: 1497.37 (exact mass); 1498.95). UV-Vis
(THF): λmax (nm)= 329, 344, 611, 680.
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 0.87 (s, 6H) ; 2.6
(s, 6H) ; 5.93 (s, 2H) ; 7.8 – 8.4 (m, 9H). 19F NMR (280 MHz,
CDCl3, 300 K): δ (ppm)= -146 (q, 1JF-B= 32 Hz, 2F). 11B NMR
(128 MHz, CDCl3, 300 K): δ (ppm)= 0.995 (t, 1JB-F= 32 Hz, 1B).
ESI-MS: m/z= 471.17 [M+Na]+, 447.18 [M-H]- (calcd for
C29H23BF2N2: 448.19 (exact mass); 448.31 (FW)). UV-Vis
(DCM) : λmax (nm)= 327, 342, 504.
Phthalocyanine-Bodipy conjugates (2c) (Sonogashira
coupling):
Pc-I4 (7) (100 mg, 0.092 mmol), the alkyne-BODIPY (6)
(100 mg, 0.36 mmol) and copper iodide (35 mg, 0.18 mmol)
were put in
a
two-necked round bottom flask. Dry
Pyrenylmethanol (4):
tetrahydrofuran (10 mL) and triethylamine (5 mL) were added,
and the resulting mixture was degased upon three series of
Pyrene-aldehyde (1 g, 4.3 mmol) and dry tetrahydrofuran
(20 mL) were placed in a round bottom flask. Sodium
borohydride (165 mg, 4.3 mmol) was added in small portions,
freeze-pump-thaw-nitrogen
refill
cycles.
Tetrakis-
(triphenylphosphine)-palladium (35 mg, 0.03 mmol) was added,
and the mixture was stirred for 18 h. Ammonium chloride was

together with small portions of methanol to help the
solubilisation (total volume of added methanol = 10 mL). An
orange solution was obtained. The reaction was quenched with a
2% concentrated hydrochloric acid solution. The solvent was
removed under reduced pressure. The white powder obtained was
dissolved in dichloromethane, washed with water and the organic
phase was dried with magnesium sulphate. The solvent was
removed under reduce pressure. The resulting solid was subjected
to silica gel column chromatography (eluent = dichloromethane).
Yield = 88%
precipitate. Pentanol was removed, and the precipitate was
resuspended in methanol and filtrated. It was subsequently rinsed
with a 1M hydrochloric acid solution until the filtrate turned
colourless, then it was rinsed with water. Yield = 75%.
1H NMR (300 MHz, d-DMSO, 300 K): δ (ppm)= 8,38 (d, 3J= 7.7
Hz, 4H) ; 8.63 (m, 4H) ; 9.13 (m, 4H). MALDI-TOF: m/z=
1080.79 [M+H]+ (Calcd for C32H12I4N8Zn : 1079.67 (exact mass);
1081.49 (FW)). UV-Vis (DMSO) : λmax (nm)= 357 ; 615 ; 682.
TMS-protected BODIPY-alkyne (8):
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 1.87 (s, 1H) ; 5.39
(s, 2H) ; 7.9 – 8.4 (m, 9H). 13C NMR {1H} (75 MHz, CDCl3, 300
K): δ (ppm)= 64,03; 123.15; 124.87; 124.93; 125.13; 125.42;
125.45; 126.14; 126.19; 127.54; 127.63; 128.07; 128.97; 130.95;
131.42; 131.44; 133.94. ESI-MS: m/z= 254.97 [M+Na]+, 214.99
[M-OH]+(Calcd. for C17H12O : : 232.09 (exact mass); 232.28
(FW)). UV-Vis (DCM) : λmax (nm)= 314 ; 327 ; 344.
Dimethylpyrrole (1 g, 10 mmol) was put in a round bottom flask
under nitrogen atmosphere, and 22 mL of dried dichloromethane
were added. The resulting mixture was sparged with nitrogen
bubbling for 90 mins. Then 3-(trimethylsilyl)-2-propynal
(0.733 mL, 5 mmol) was added drop wise at -70°C over a period
of 30 min. The solution became purple. After the addition,
stirring was carried on for 15 more minutes and removed. The
solution was allowed to come to room temperature during 3 h.
The solution became orange. First, p-Chloranil (1.23 g, 10 mmol)
was added, the reaction mixture was stirred for 1 h until it turned
purple. Then, triethylamine (6.74 mL, 50 mmol) was added, the
reaction was stirred for 30 min at room temperature. Finally,
boron trifluoride diethyl etherate was added drop wise and the
reaction was stirred for 16 h. Dichloromethane was added to the
mixture and the organic phase was extracted with
dichloromethane, washed with water and dried over magnesium
sulphate. The solvent was removed under reduced pressure.
Acetone was added until there was no more smoke emission, and
was removed under reduced pressure. The solid obtained was
subjected to column chromatography (silica gel, using a
petroleum ether: dichloromethane mixture (70 : 30 vol.) as the
eluent). Yield = 70%.
4-(pyren-1-ylmethoxy) phthalonitrile (5):
4-Nitrophthalonitrile (87 mg, 0.5 mmol) and 1-pyrenylmethanol
(4) (116 mg, 0.5 mmol) were put in a round bottom flask.
Dimethylsulfoxyde (10 mL) was added and the mixture was
stirred until the powder was completely dissolved. Potassium
carbonate (250 mg, 1.75 mmol) was added in small portions over
a period of 2 h. The reaction mixture was stirred for 22h at room
temperature. Then it was poured in 50 mL of water that results in
the precipitation of a white solid. The solid was isolated by
filtration on a Büchner, then rinsed with water, heptane and
diethyl ether. Yield = 59%.
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 5.80 (s, 2H) ; 7.24
3
3
(s, 1H) ; 7.34 (d, J= 8.7 Hz, 1H) ; 7.68 (d, J= 8.7 Hz, 1H) ; 8 –
8.25 (m, 9H). ESI-MS: m/z= 356.98 [M-H]-, 381.08 [M+Na]+
(Calcd for C25H14N2O: 358.11 (exact mass); 358.39). UV-Vis
(DCM) : λmax (nm)= 315 ; 329 ; 346.
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 0,27 (s, 9H) ; 2.5
(s, 6H) ; 2.4 (s, 6H) ; 6.04 (s, 2H). 13C NMR {1H} (75 MHz,
CDCl3, 300 K): δ (ppm)= 0.56; 14.73; 15.65; 100.62; 115.11;
120.20; 120.91; 133.21; 142.37; 154.55. ESI-MS: m/z= 345
[M+H]+; 325 [M-F]+; 367 [M+Na]+. (calcd for C18H23BF2N2Si:
344.17 (exact mass); 344.28 (FW))
TMS-deprotected BODIPY alkyne (6):
TMS-protected BODIPY-alkyne (8) (400 mg, 0.999 mmol) was
dissolved in 80 mL of methanol. Potassium fluoride (290 mg,
4.995 mmol) was added and the solution was stirred for 90 min.
The solvent was removed under reduced pressure and the powder
was dissolved in dichloromethane. The organic phase was
extracted with dichloromethane, washed with water and dried
over magnesium sulphate. The solvent was removed under
reduced pressure to afford a brown powder. Yield = 70%
4-Iodo-phthalonitrile (9):
4-amino-phthalonitrile was placed in a three-necked round
bottom flask. 50 g of crushed ice and 80 mL of hydrochloric acid
(37%) were added. Then a solution of sodium nitrite (3 mg, 43
mmol) in water (30 mL) was slowly added to the reaction
mixture at 5°C over 30 min. The reaction mixture was stirred at
5°C for 1h. The precipitate formed was filtrated and washed with
water (50 mL). The filtrate was added dropwise on a potassium
iodide solution (7 mg, 42 mmol) in 30 mL of water at 5°C over a
period of 1 h. The reaction mixture was warmed to room
temperature during 14 h. An orange powder was filtrated on
Büchner, and dissolved in toluene (affording an orange filtrate).
The organic phase was washed with saturated bicarbonate
sodium solution, saturated sodium thionite solution, brine, then
dried over magnesium sulphate. After concentration under
reduced pressure, the resulting solid was subjected to column
chromatography (silica gel, eluent = dichloromethane: toluene,
50: 50 vol.). Yield= 51%.
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 2.43 (s, 6H) ; 2.5
(s, 6H) ; 3.89 (s, 1H) ; 6.04 (s, 2H). 13C NMR {1H} (75 MHz,
CDCl3, 300 K): δ (ppm)= 14.77; 15.60; 79.37; 94.52; 119.06;
121.19; 133.46; 142.75; 155.23. 19F NMR (280 MHz, CDCl3, 300
K): -146 ppm (q, 1JF-B= 65.5, 32.7 Hz, 2F). ESI-MS: m/z= 273.04
[M+H]+, 295.01 [M+Na]+. (calcd for C15H15BF2N2: 272.13 (exact
mass); 272.10 (FW)). UV-Vis (DCM) : λmax (nm)= 338, 509, 544.
[2,9(10),16(17),23(24)-tetraiodophthalocyaninato]zinc (Pc-I4)
(7):
4-Iodo-phthalonitrile
(9)
(1 g,
3.95 mmol),
1,8-
diazabicyclo{5.4.0}undec-7-ene (602 mg, 3.95 mmol) and zinc
acetate (180 mg, 0.98 mmol) were put in a round bottom flask
and 10 mL of pentanol were added. The mixture was heated at
130°C and turned blue. After 23 h of reflux, the solution was
allowed to come to room temperature affording a dark
1H NMR (300 MHz, d-DMSO, 300 K): δ (ppm)= 7.85 (d, 3J= 7.8
Hz, 1H); 8.31 (dd, 3J= 7.8, 1.6 Hz, 1H) ; 8.58 (d, 3J= 1.6 Hz, 1H).
13C NMR {1H} (75 MHz, CDCl3, 300 K): δ (ppm)= 99.78;
113.95; 115.05; 115.22; 117.16; 134.12; 142.18; 142.59. ESI-

MS: m/z= 276.81 [M+Na]+; 252.83 [M-H]-; (calcd for C8H3IN2 :
253.93 (exact mass); 254.03 (FW)).
suspension was used immediately.
2.3.5. Imaging studies
BODIPY-dicyanobenzene conjugate (14)
Upon fixation (using methanol) cells were imaged using a
biphoton microscope and then a confocal microscope.
4-Iodo-phthalonitrile (9) (55.6 mg, 0.22 mmol), the alkyne-
BODIPY (6) (60 mg, 0.22 mmol) and copper iodide (6,9 mg,
0.036 mmol) were put in a two-necked round bottom flask. Dry
tetrahydrofuran (10 mL) and triethylamine (4 mL) were added,
and the resulting mixture was degased upon three series of
3. Results and Discussions
3.1. Synthesis and Characterization of probes
freeze-pump-thaw-nitrogen
refill
cycles.
Tetrakis-
(triphenylphosphine)-palladium (11,5 mg, 0.01 mmol) was
added, and the mixture was stirred for 22 h. Ammonium chloride
was added to quench the reaction, the organic phase was
extracted with dichloromethane, washed with water and with a
saturated solution of ammonium chloride, dried over MgSO4,
filtered, and the solvents were removed under reduced pressure.
The resulting residue was subjected to column chromatography
on silica gel using a dichloromethane-petroleum ether mixture
(4:1 vol.) as the eluent. Yield = 60%
The syntheses of pyrene-containing probes 1 and 3 were the most
straightforward to carry out. Pyrene-Pc 1 was synthesized by
reduction of pyrene carboxaldehyde with NaBH4 to afford
pyrenyl
carbinol
4
that
undergoes
SNAr
with
nitrodicyanobenzene to afford pyrenyloxydicyanonobenzene 5.
The latter was subsequently cyclotetramerized in the presence of
zinc salt and DBU to afford target 1, because of solubility issues
1
it was only characterized by HRMS and UV/Vis. Moreover, H-
NMR analysis allowed to identify the presence of key signals
(albeit very broad). The Pyrene-BODIPY conjugate 3 was
synthesized in a three-steps one-pot procedure starting from a
1H NMR (300 MHz, CDCl3, 300 K): δ (ppm)= 2.49 (s, 6H) ; 2.54
(s, 6H) ; 6.10 (s, 2H), 7.84 (m, 3H). 19F NMR (280 MHz, CDCl3,
TFA-catalyzed
dimethylpyrrole,
condensation
followed
of pyrenyl-carbinol
by p-chloranil oxidation,
and
1
300 K): -145.9 ppm (q, JF-B= 65.5, 32.7 Hz, 2F). ESI-MS: m/z=
273.04 [M+H]+, 295.01 [M+Na]+. (calcd for C23H17BF2N4: (exact
mass); 397.02 [M-H], 380.13 [M-F]+ (FW)).
deprotonation with triethylamine followed by borylation with
BF3-etherate. Target 3 was characterized by 1H-NMR, 13C-NMR,
11B-NMR (doublet at ca. 1 ppm), 19F-NMR (doublet of triplet at -
146 ppm) and UV/Vis (characteristic absorption band at 327, nm
and 342nm and 504).
2.3.2. Fluorescence studies
Fluorescence measurements were performed on a Jasco FP-8500
spectrofluorometer equipped with a Xe source. Excitation was
performed at 329 nm (1a), 551 nm (2c), and 328 nm (3a).
Emission spectra were recorded for an absorbance at 329 nm
(pyrene), 551 nm (BODIPY) comprised between 0.03 and 0.07.
Scheme 1. Synthesis of pyrene-containing targets (1) and (3) (i) NaBH4,
THF, MeOH, yield: 88% ; (ii) K2CO3, DMSO, yield: 59% ; (iii) Zn(OAc)2,
DBU, pentanol, yield: 17% ; (iv) a) dimethylpyrrole, TFA, b) p-chloranil, c)
NEt3, d) BF3.Et2O, yield: 22%.
2.3.3. Cell Culture and Fixation
B16F10 melanoma cells were grown in RPMI supplemented with
10% fetal calf serum and 1% streptomycin and penicillin. Two
days before imaging studies, cells were harvested and placed in
8-well microscopy chambers (8 chambers polystyrene vessel
(with tissue culture treated glass slides; BD Falcon Culture slides
Ref 3541 18)). Cells were incubated with non-supplemented
RPMI mixed with probes 1,2 in solution in DMSO (the % of
which, upon addition in the well, does not exceed 1,5%) or probe
3 as a liposomal suspension. Upon 1 h incubation, the medium
was removed and cells were rinsed with PBS. Then cold
methanol (stored at -30°C) was added in each well, and the plates
chambers were stored for 5 min at 4°C in the fridge. Methanol
was then carefully removed from the well, and replaced with cold
PBS (4°C) and the plate chambers were stored on ice up to the
biphoton microscope. Then the walls were removed, PBS was
added on the cells, and a microscopy glass was added on the cells
prior to microscopy study.
Pc-BODIPY 2c was synthesized from alkynyl-BODIPY 6
(scheme 2A) and tetraiodophthalocyanine ZnPCI4 7 (scheme 2B).
The condensation of dimethylpyrrole and trimethylsilylpropynal
led to the formation of dipyrromethane that was oxidized,
deprotonated and borylated to afford 8 in an overall three-steps
one pot procedure. It should be noted that no acid catalyst was
necessary in the synthesis of BODIPY 6, unlike that of BODIPY
3 (scheme 1 and 2). The protective group in 8 was removed to
afford unprotected alkynyl-BODIPY 6. Tetraiodophthalocyanine
synthon 7 was synthesized from aminodicyanobenzene as
follows: the latter was reacted with sodium nitrite to afford
intermediate diazonium salt (none isolated), which reacts with
iodide to afford iododicyanobenzene 9. DBU-catalyzed
cyclotetramerization of the latter in refluxing pentanol afforded
ZnPcI4 synthon 7 (Scheme 2B). This synthon was obtained as a
mixture of regioisomers that cannot be separated. Subsequent
Sonogashira coupling (Cu(I) / Pd(0) catalyzed) between
iodinated phthalocyanine 7 and alkynyl-BODIPY 6 afforded
BODIPY-Pc conjugate 2c. It was obtained as a mixture with
various degree of fonctionnalisation (from 1 to 4 BODIPY unit
per Pc as determined by MALDI-TOF MS) because of
incomplete Sonogashira coupling. The mixture was purified by a
series of washings with methanol to remove impurities. It was
eventually subjected to Sonogashira coupling again, which
increased the conjugation rate in BODIPY. Several attempts to
separate the different BODIPY-Pc conjugates by standard
chromatography on silica failed.
2.3.4. Preparation of Liposomes
Liposomes were prepared by the injection method following
earlier procedures [3b,]. Two solutions have been prepared : 1,2-
dipalmitoyl-sn- glycero-3-phosphocholine (14.6 mg) in ethanol
(1 mL) (solution 1), and the probe (5 μmol) in chloroform
(10 mL) (solution 2). 100 µmol of each solution were mixed and
quickly injected using an Hamilton seringe into a vigorously
stirred PBS (10 mL) at 60°C. Stirring at 60°C went on for 2 min,
then heating was stopped and the solution was gradually cooled
down to room temperature under stirring. The liposome

A
B
C
ICP: B/Zn = 2.5
- 146 ppm
D
0.524 ppm
dB (ppm)
dF (ppm)
E
Scheme 2. Synthesis of synthons, such as BODIPY 6 and ZnPcI4 7 and
subsequent Sonogashira coupling between 6 and 7 leading to target 2c. (i) p-
chloranil, NEt3, BF3.Et2O, yield: 70%; (ii) KF, MeOH, yield: 70 % (iii)
NaNO2, KI, CH3OH, yield: 51%; (iv) DBU, Zn(OAc)2, pentanol, yield in 7:
75%; (v) 6, Pd(PPh3)4, CuI, Et3N, THF, yield in 2c: 60%; yield in 14 : 40%.
Fig. 2. Characterization of 2c: A. UV/Vis in non-coordinating solvent
(dichloromethane, black) and in the presence of coordinating pyridine (red);
B: ICP-AES; B: Mass Spectrometry; C: 19F-NMR; D: 11B-NMR; E: 1H-NMR.
Attempts to couple up to four BODIPY were made : (i) the
isolated statistical mixture was subjected again to Sonogashira
coupling up to three times. Upon removal of non-phthalocyanine
components by washings, ICP and 1H-NMR measurement
indicated a slight improvement, with a ratio close to 1:4 (i.e. four
BODIPY per Pc). (ii) Finally, another synthetic approach
The characterization of the statistical mixture in 2c was carefully
achieved in light of the spectroscopic signatures of its two
components (BODIPY 6 and Pc 7). It was achieved using an
array of spectroscopic tools such as HRMS, UV-Vis, 1H, 11B and
19F NMR and also ICP (Figure 2 A-E). They offered a series of
spectroscopic handles that allowed to conclude about the nature
of 2c. (a) First, upon extensive extractions and washings, TLC of
2c shows only one purple spot on TLC (with a short tail),
indicative of the removal of non-phthalocyanine and non-
BODIPY materials. (b) MALDI-TOF mass spectrometry shows
the molecular peaks of species that correspond to phthalocyanine
bearing two to four BODIPY moieties. The corresponding peaks
with loss of fluorine atoms were also observed. (c) ICP analysis
gave an Zn/B ratio corresponding to about 2.5 Boron atoms
(from the BODIPY) per atom of Zinc (from the Pc). (d) 1H NMR
of 2c is not as resolved as that of phthalocyanine precursor 7.
However, it clearly shows the protons of the phthalocyanine
isoindolic moieties (7 - 8 ppm) and the protons of the BODIPY
moieties, such as pyrrolic protons (6 - 6,5 ppm), and methyl
protons (3 ppm). The integration of these two signals roughly
concurs with the BODIPY/Pc ratio determined by ICP. Note that
the broad signals may be the result from stacking and the
presence of multiple regioisomers. (e) The proof of the presence
of the BODIPY moiety was also shown by 11B NMR and 19F
NMR signals at δ (ppm)= 0.524 et δ (ppm)= -146 ppm,
respectively. (f) UV-Vis spectroscopy performed in
dichloromethane shows three bands that correspond to the Pc
moiety at 379 nm (Soret) and 689 nm (Q bands), and that of the
BODIPY moiety at 379 nm and 555 nm. Not only all bands
appear to have a broad shape, but also the Q bands appear flat.
Upon addition of a coordinating solvent in the solution, such as
pyridine (in red) or tetrahydrofuran, the intensity of Q bands
increased significantly with respect to that of other bands.
Moreover, a bathochromic shift was observed. These data are the
signature of a natural aggregation phenomenon that is inhibited
upon addition of coordinating molecules preventing the
molecular stacking. Altogether, these spectroscopic data indicate
that 2c is a statistical mixture that corresponds to the grafting of
about 2.5 BODIPY per Pc, where the final molecular construct
indicates a strong tendency to aggregate in non-coordinating
solvents.
consisted in coupling alkynyl-BODIPY
6
with the
iodophthalonitrile precursor first to afford BODIPY-
9
phthalonitrile 14. Subsequent cyclotetramerization of 14 did not
work in our hands, unlike what Durmus reported with an
analogous system (6).
3.2. Absorption and Fluorescence emission studies
The overlay of the absorbance spectra of Pc-pyrene 1 and its
precursor pyrenyl-methanol 4 and Pc 7 shows a good match
between their two maximum absorption bands : bands at 329 nm
and 345 nm correspond to the pyrene, whereas bands at 611 nm
and 678 nm correspond to the Pc (Fig. 3A). Fluorescence
emission spectra obtained upon excitation at 329 nm show that
the bands corresponding to pyrene are no longer present, whereas
that corresponding to Pc are present (Fig. 3B). This indicates that
the energy transfer occuring from pyrene to Pc occurs, as
previously described by Ozcesmeci et al.
Fluorescence emission
Absorbance (a.u.)
B
A
Wavelength (nm)
Wavelength (nm)
Fig. 3. Pc-pyrene 1. A : Absorption spectra of 1-pyrenylmethanol (4) (yellow)
in DCM, Pc-I4 (7) (blue) in DMSO and (1) (dotted green) in DMSO. B : Pc-
pyrene 1 emission spectrum (red) and excitation spectrum (purple) measured
in DMSO (λ ex = 329 nm; λ em = nm)
The overlay of the absorbance spectra of Pc-BODIPY 2c and that
of its two components BODIPY 6 and Pc 7 shows a good match
between their maximas, which appear at 373 and 551 nm with

BODIPY (6), and 373 and 694 nm with Pc 7 (Fig. 4A). Upon
excitation at 551 nm (BODIPY), subsequent fluorescence
emission spectra show that the transfer from the BODIPY
antenna towards the Pc platform occurred (Fig. 4B). Indeed,
emission peak corresponding to the maximum emission of Pc is
observed (700 nm). Moreover, when the excitation was achieved
at 373 nm subsequent fluorescence emission also occured leading
to a similar emission spectrum.
failed to reach a successful incorporation rate with Pc-containing
species 1-2 (for lack of solubility reasons in solvent classically
used for liposomes, Supp. Info Fig S9). Hence, the latter were
used as a solution in DMSO and subsequently injected in RPMI
such as DMSO content does not reach more than 1%. Controls,
i.e. untreated cells that do not contain any probe (raw 1, Figure 6)
barely fluoresce upon two-photon excitation at various
wavelengths and whatever filter was used (blue, green, red
channels). In these images, it should be noted that the
background remained dark whereas stained cells are well defined.
Staining of membranes and cytoplasm occurred, whereas the
nucleus remains unstained.
Fluorescence emission
B
A
Absorbance (a.u.)
DAPI (blue channel:
400-492 nm)
DIO (green channel:
500-550 nm)
QDot655 (red channel:
601-657 nm)
Controls
C
A
B
Wavelength (nm)
Wavelength (nm)
Fig 4. BODIPY-Pc 2c. A : Absorption spectra of BODIPY-alkyne 6 (pink,
measured in DCM), phthalocyanine precursor Pc-I4 7 (blue, measured in
DMSO) and phthalocyanine-BODIPY conjugate 2c (dotted green, measured
in THF). B : BODIPY-Pc 2c emission spectrum (red, λ ex= 551 nm) and
excitation spectrum (purple) measured in THF (λ em = nm) .
Pc – BODIPY 2a
The overlay of the absorbance spectra of pyrene-BODIPY 3 with
that of its components, pyrene and BODIPY, shows two
absorption maxima at 328 and 343 nm for the pyrenyl
component, and a maximum at 504 nm that corresponds to the
BODIPY. Fluorescence emission spectrum of 3 shows a single
band at 515 nm that corresponds to the emission of BODIPY. It
also indicates that there is a complete transfer from the pyrene
moiety to the BODIPY as no band was found below 500 nm (Fig.
5). Hence, there is no more fluorescence signal corresponding to
pyrene.
F
D
E
Pyrene – BODIPY 3
C
G
H
Absorbance (a.u.)
Fig 6. Fluorescence biphoton microscopy images (exc. 750 nm; channels:
blue (A,D,G), green (B, E, H) and red (C, F)) of B16-F10 melanoma cells
upon treatment and fixation with methanol: untreated cells (controls, images
A-C), and cells incubated with 1 M of probes: probe Pc-BODIPY 2c
(solution in RPMI with 1.5% DMSO, images D-F) and Pyrene-BODIPY 3
(liposome suspension, images G-H).
Wavelength (nm)
Fig. 5. BODIPY-pyrene 3. Absorption spectrum (dotted green), emission
spectrum (red) and excitation spectrum (purple) measured in DCM (λex
328 nm; λ ex = 515 nm)
=
This is unlike cells treated with Pc-BODIPY conjugate 2c (raw 2,
Figure 6) in solution in RPMI with 1% DMSO: strong
fluorescence emission was observed mostly in the green canal (E,
BODIPY emission) but also in the blue (D) and red canal (F).
Cells treated with Pc-Pyrene conjugate 1 (in solution with 1%
DMSO) are fluorescent with fluorescence emission mostly in the
blue (pyrene) and red (Pc) windows (data not shown, see Fig S9
in the SI). Interestingly, cells treated with a liposomal solution of
BODIPY-pyrene conjugate 3 emit in the blue (pyrene, G) and
green (BODIPY, H) windows and barely in the red. These results
were revisited using confocal microscopy (Figure 7): it also
indicates that cells were stained with fluorescent probes 2c,
results with different canals also indicate that transfers from one
antenna to the other occurred and may be reminiscent of the
spectrofluorimetry studies (fluorescence emission spectra Figures
3-5). Altogether microscopy and spectrofluorimetry studies seem
to indicate that transfers from the donor to the acceptor moiety
within a fluorophore conjugate do occur. However, these two
studies do not seem to be a mirror image of each other: upon
Table 1. Absorption and emission data.
Compound
Solvent
Abs. max. (nm)
329, 345
Em. Max. (nm)
Pyrene-OH (4)
PcI4 (7)
DCM
355
690
700
515
515
700
DMSO
DMSO
DCM
373, 611, 678
373, 694
Pc-Pyrene4 (1a)
BODIPY-CCH (6)
BODIPY-Pyrene (3a)
Pc-BODIPY4 (2a)
360, 551
DMSO
DCM
345, 551
373, 551, 694
3.3. Cellular Imaging studies
Cell imaging studies were carried out in light of previous reports
using BODIPY, Pc, pyrene monomeric probes [2b, 11]. Here,
prior entrapment of fluorescent probes 1-3 in SUV liposomes
was attempted following our earlier procedure [3b,10]. It was
successful with BODIPY-pyrene conjugate 3 (Fig. 6) but we

excitation at (750-800 nm) subsequent cell imaging remain
possible in the green canal indicating emission of the BODIPY
emission, which spectrofluorimetry studies showed does not
occur. Such differences may possibly be explained from various
environnement: DMSO (spectrofluorimetry) vs cellular
environnement (microscopy studies).
UV-Vis: UltraViolet-Visible
DMSO: Dimethyl sulfoxyde
DCM: Dichloromethane
CDCl3: deuterated chloroform
THF: Tetrahydrofuran
PBS: Phosphate Buffered Saline
RPMI: « Roswell Park Memorial Institute » medium
TMS: TriMethylSilyl
ICP-AES: Inductively Coupled Plasma - Atomic Emission
Spectrometry
A
B
C
D
LC-MS: Liquid Chromatography – Mass Spectrometry
HPLC: High Performance Liquid Chromatography
MALDI-TOF: Matrix-Assisted Laser Desorption/Ionisation -
Time of Flight
Fig 7. Confocal images of B16F10 cells incubated with a solution of 2c (1
M) in solution (excitation at 405 nm) : transmission (A), fluorescence with
collection at 400-500 nm (B) or 668 – 678 nm (C), and superposition (D).
ESI-MS: Electrospray Ionization Mass Spectrum
References and notes
4. Conclusion
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nm), pyrene-BODIPY 3 (ca. 300-500 nm), and BODIPY-
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Pc: Phthalocyanine
BODIPY : BOron DIPYromethene
NMR: Nuclear Magnetic Resonance

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