Synthesis and characterization of glycoconjugated porphyrin triphenylamine hybrids for targeted two-photon photodynamic therapy

Article abstract of DOI:10.1021/jo402658h

In order to avoid side effects at the time of cancer eradication to the patients, the selectivity of treatments has become of strategic importance. In the case of photodynamic therapy (PDT), two-photon absorption combined with active targeting of tumors c

Full text of DOI:10.1021/jo402658h

Article
pubs.acs.org/joc
Synthesis and Characterization of Glycoconjugated Porphyrin
Triphenylamine Hybrids for Targeted Two-Photon Photodynamic
Therapy
Fabien Hammerer,†,‡,§,∥ Guillaume Garcia,†,‡,§,∥ Su Chen,†,‡,§,∥ Florent Poyer,†,§,∥,⊥ Sylvain Achelle,†,‡,§,∥,$
Celine Fiorini-Debuisschert,# Marie-Paule Teulade-Fichou,†,‡,§,∥ and Philippe Maillard*,†,‡,§,∥
́
†Institut Curie, Section de Recherches, Bat
̂
110-112, Centre Universitaire, F-91405 Orsay, France
110, Centre Universitaire, F-91405 Orsay, France
Paris-Sud, Centre Universitaire, F-91405 Orsay, France
∥CNRS GDR 3049 PHOTOMED, UMR 5623, Universite
Paul Sabatier, F 31062 Toulouse Cedex 9, France
‡UMR 176 CNRS, Bat
§Universite
̂
́
́
⊥U759 INSERM, Bat
̂
112, Centre Universitaire, F-91405 Orsay, France
#CEA Saclay DSM-IRAMIS/SPCSI - Lab. Nanophotonique, F-91191 Gif-sur-Yvette Cedex, France
S
* Supporting Information
ABSTRACT: In order to avoid side effects at the time of
cancer eradication to the patients, the selectivity of treatments
has become of strategic importance. In the case of photo-
dynamic therapy (PDT), two-photon absorption combined
with active targeting of tumors could allow both spatial and
chemical selectivity. In this context, we present the synthesis,
spectroscopic, and biological properties of a series of
porphyrin-triphenylamine hybrids with excellent singlet oxy-
gen production capacities and good two-photon absorption.
should allow also greater precision than is achievable by
INTRODUCTION
■
conventional one-photon excitation as a consequence of the
quadratic dependence of two-photon excitation on the local
light intensity.7 Simultaneous two-photon excitation of
porphyrin derivatives can be carried out within the biological
optical window. Because classical porphyrin photosensitizers
exhibit small 2PA values using femtosecond pulsed lasers (less
than 50 GM where 1 GM = 10−50 cm4·s·molecules−1),8,9
molecular engineering is necessary to improve the 2PA
properties unless it becomes necessary to use very high laser
intensities that can be deleterious for the biological tissues.10,11
The directions that have been explored consist of more
extended cyclic polypyrroles,12,13 dimers, or oligomers of
conjugated porphyrins14−18 or the use of 2PA light-harvesting
antenna.19−21 Spangler and co-workers have developed
porphyrin−triphenylamine hybrids for 2PA−PDT.22−24 In
these structures, a cooperative effect has been demonstrated:
these compounds exhibit interesting 2PA cross sections
superior to the sum of the cross sections of the two parts
taken separately. Spangler showed that, in these compounds, π-
conjugation between the porphyrin and the triphenylamine part
is necessary to obtain interesting 2PA properties. Moreover,
these structures possess a high singlet oxygen quantum yield.
Photodynamic therapy (PDT) is an emerging treatment
designed to eliminate diseased tissues such as malignant
tumors.1−4 It involves a photosensitizing molecule (PS) that
can be activated to its triplet excited state and transfers its
energy to molecular triplet oxygen resulting in the generation of
singlet oxygen or other reactive oxygen species (ROS). These
highly reactive species lead to the destruction of targeted
diseased tissues by biochemical damage. A major advantage of
PDT in comparison with other treatments such as chemo- or
radiotherapy is that, in the absence of light activation, the PS is
well-tolerated by the cells without cytotoxic effect. Therefore,
PDT has the potential to selectively destroy malignant cells
while sparing the nonirradiated normal tissues.
PS currently used in PDT are mainly cyclic tetrapyrroles or
porphynoids because of their long excited triplet-state lifetime.5
These PS in free-base form have five one-photon absorption
(1PA) bands in the visible wavelength range (400−700 nm),
and despite promising results, an important limitation arises
from the fact that the penetration depth of these wavelengths is
confined near the surface of the tissues. However, the
absorption is much lower in the optical window of biological
tissues between 700 and 1300 nm to obtain an effective
photobiological effect.6 One way to overcome this problem
consists of taking advantage of two-photon absorption (2PA)
processes. In this nonlinear optical phenomenon, two photons
of lower energy are absorbed simultaneously. 2PA−PDT
Received: December 20, 2013
Published: January 16, 2014
© 2014 American Chemical Society
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Figure 1. Structure of target compounds.
a
Another limitation of PDT is the low selectivity and
specificity of the PS for tumor cell: high drug and light doses
are thus required to compensate forthis, leading to damage of
healthy tissues. Active targeting of membrane receptors
represents an obvious improvement.25−28 It has been reported
that lectin-type receptors are overexpressed in certain malignant
cells29−32 and that carbohydrates such as α-mannose and β-
galactose have specific interaction with these receptors.33−35
Over the past decade, we have focused our efforts on the
preparation and in vitro and in vivo evaluation of the 1PA
phototoxicity of glycoconjugated tetrapyrrolic macrocycles.36
Recently, our group has developed a series of glycoconjugated
vectorized porphyrin oligomers for 2PA PDT.37−39
Scheme 1. Synthesis of Triphenylamine Residues
Following the strategy used by Spangler, we have developed
a new design of porphyrin−triphenylamine photosensitizer for
2PA PDT exploiting a branching effect. The aim of this work is
to describe the synthesis, photophysical (1- and 2PA), and
photobiological properties (1PA) of a series of carbohydrate-
vectorized porphyrin−triphenylamine hybrids 1−5 (Figure 1).
RESULTS AND DISCUSSION
■
a
Key: (i) DMF, POCl3, 95 °C, 4 h; (ii) Br2, CH2Cl2, reflux, 4 h; (iii)
I2, Ag2SO4, EtOH, rt, 15 h; (iv) PyrCH2PO(OEt)2, NaH, THF, rt, 48
h; (v) X = I, TMSA, Pd(PPh3)2Cl2, CuI, THF, Et3N, rt, 15 h; (vi)
TBAF, CH2Cl2, 0 °C, 30 min.
Synthesis. Triphenylamine was converted into the diacy-
lated compound 6 according to a previously described
Vilsmeier−Haack procedure using POCl3 and DMF with 58%
yield (Scheme 1).40 The third phenyl ring was halogenated by
action of bromine leading to compound 7a (86% yield)41 or
with iodine and silver sulfate to afford 7b in quantitative yield.
Horner−Wadsworth−Emmons (HWE) reaction of 7a with (4-
pyridinyl)methyl diethyl phosphonate [PyrCH2PO(OEt)2] led
to compound 8a with trans-geometry (see the NMR results:
10. It should be noted that Sonogashira coupling of bromo
derivative 7a was not successful.
5-Bromo-10,15,20-triphenylporphyrinatozinc(II) obtained
according to Senge’s strategy43 was converted to silylated
compound 11 via Sonogashira coupling in 89% yield. The
synthesis of the target compounds 1−3 via Sonogashira cross-
coupling was first considered (Scheme 2), taking into account
the fact that such cross coupling of 5,10,15-triphenyl-20-
(trimethylsilyl)ethynylporphyrinatozinc(II) 11 has previously
been described.1,8 However, reaction of 11 with triphenylamine
derivative 8a led to the homocoupling product and desired
compound 1 in very small yield (19%) (Scheme 2). An
3
coupling constant JH−H′ = 16 Hz characteristic of E
configuration, 46% yield) according to the procedure previously
developed in our group.42
Sonogashira cross-coupling of iodo derivative 7b with
trimethylsilylacetylene (TMSA) gave silylated compound 9
(91% yield), which was deprotected quantitatively by action of
tetrabutylammonium fluoride (TBAF), leading to compound
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a
Scheme 2. Synthesis of Compounds 1−3
a
Key: (i) TMSA, Pd(PPh3)2Cl2, CuI, THF, Et3N, rt, 15 h; (ii) TBAF, CH2Cl2, 0 °C, 30 min then Pd2(dba)3, AsPh3, THF, Et3N, reflux, 18 h; (iii) 10,
Pd(PPh3)2Cl2, CuI, THF, Et3N, rt, 20 h; (iv) TFA, CHCl3; (v) PyrCH2PO(OEt)2, NaH, THF, rt, 48 h; (vi) BzimCH2PPh3Cl, DBU, THF, rt, 15 h.
alternative strategy was thus adopted, involving compound 12
which was obtained from 10 and 5-bromo-10,15,20-
trisphenylporphyrinatozinc(II) by Sonogashira coupling reac-
tion (84% yield). The zinc atom was removed by action of
trifluoroacetic acid (TFA), leading to the free base analogue 13
(63% yield). Compounds 12 and 13 were then converted to
target compounds 1 and 2 by HWE reaction (with yields of 84
and 26%, respectively, exclusively in E configuration as was
confirmed by 1H NMR with a coupling constant 3JH−H′ of about
16 Hz for the alkenyl hydrogens). Wittig reaction between 12
and (N-methylbenzimidazolyl)methyltriphenylphosphonium
chloride (BzimCH2PPh3Cl) in the presence of 1,8-diazabicy-
cloundec-7-ene (DBU) afforded the compound 3 in 22% yield.
The second-generation compound 4 decorated with water-
solubilizing TEG fragments has also been designed (Scheme 3).
5,10,15-Tris(4-hydroxyphenyl)porphyrin 15 was obtained by
the deprotection of isopropoxy groups with BBr3 of 5,10,15-
tris(4-isopropyloxyphenyl)porphyrin.43,44 Bromination with N-
bromosuccinimide (NBS) (compound 16) and metalation with
zinc(II) acetate afforded compound 17 with 81% yield over two
steps. Then, 2-[2-(2-methoxyethoy)ethoxy]ethanol tosylate 14
and 17 reacted through a Williamson coupling reaction leading
to 18 with 88% yield. Sonogashira cross-coupling reaction with
TMSA afforded compound 19 with 69% yield. Compound 19
was deprotected with TBAF and coupled with 7b to afford 20
(95% yield), which was engaged in the HWE reaction with
PyrCH2PO(OEt)2 giving compound 4 with 99% yield (E
geometry: see NMR results, characteristic coupling constant
3JH−H′ ≈ 16 Hz).
The synthesis of a third-generation compound bearing α-
mannose targeting moieties is described in Scheme 4. The
Williamson’s reaction between 15 and 2-bromoethoxyethoxy-
O-2′,3′,4′,6′-tetraacetyl-α-D-mannose, previously described by
our team,45 led to compound 21 which is converted in three
steps in 22.36 Unfortunately, the Sonogashira cross-coupling
reaction46 does not afford the desired diacyl intermediary 25
and could not be optimized.
Another synthetic pathway was tested. Direct meso-
functionalization of 15 led to 23 (48% over three steps),
whose Sonogashira coupling with 7b was efficient leading to
compound 24 with a 69% yield. The glycosyl moieties were
added on compound 24 by Williamson’s reaction to afford
compound 25 (49%). Unfortunately, the pyridinyl groups
could not be introduced on 25 to obtain compound 30. The
same synthetic problems were encountered to convert
compound 24 in substrate 29 due to the deprotonation of
the phenol groups inducing the precipitation of the substrate.
A third synthetic strategy was envisaged: Tris-isopropox-
yphenylporphyrin was brominated on its free meso position,
metalated with zinc salt, and coupled to TMSA to afford
compound 26 in a yield of 78% over three steps. Sonogashira
coupling reaction with 7b led to 27 (19%), and a HWE
reaction, directly followed by the deprotection of the alkoxy
groups, afforded 29 as a free base analogue (12% over two
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a
Scheme 3. Synthesis of Compound 4
a
Key: (i) NBS, CHCl3, pyridine, 0 °C, 30 min; (ii) Zn(OAc)2, CHCl3, MeOH, reflux, 5 min; (iii) Cs2CO3, DMF, rt, 18 h; (iv) TMSA,
Pd(PPh3)2Cl2, CuI, THF, Et3N, rt, 4 h; (v) TBAF, CH2Cl2, 0 °C, 30 min then 7b, Pd2(dba)3, AsPh3, THF, Et3N, rt, 20 h; (vi) PyrCH2PO(OEt)2,
NaH, THF, rt, 48 h.
steps, E configuration). Compound 29 reacted with the bromo-
diethylene glycol protected mannosyl compound to afford 30
(20%). Compound 30 was metalated under the usual
conditions, and its glycosyl moieties were deprotected by
hypothesis is illustrated in Figure 4, which presents emission
spectra of 1, 8b, and 12 at the same concentration following
excitation at 380 nm. Almost total quenching of the
triphenylamine is observed in the hybrid as well as increased
porphyrin emission in the hybrid. Similar results (absorption
and emission) were obtained for compounds 4 and 5 (see the
Supporting Information). All compounds display relatively
weak fluorescence emission but excellent photosensitizing
properties (ΦΔ 0.64 to 0.84) in a polar solvent like DMF.
Characterization of the two-photon absorption properties of
compounds 1−5 was obtained after two-photon-induced
fluorescence (TPIF) measurements, with a femtosecond
Ti:sapphire laser source delivering 100 fs pulses at 76 MHz
repetition rate over the spectral range from 770 to 900 nm. The
TPIF intensities of the samples were measured relative to a
solution of fluorescein, the ratio of the fluorescent signals
enabling further determination of the 2PA cross section (δ)
assuming equal one- and two-photon fluorescence quantum
yields.49 The dependence of the TPIF intensity with the power
of the incident exciting IR beam was systematically checked, a
quadratic dependence being observed only for exciting
wavelengths above 830 nm. Oppositely, a linear variation of
the fluorescence signal intensity was observed for exciting
wavelength below this value, pointing to a one-photon excited
fluorescence process. The latter was attributed to hot-band
absorption as previously reported for porphyrin oligomers or
hybrids.50 The 2PA cross-section of every compound was thus
determined at λexc = 830 nm; this wavelength led to optimized
two-photon fluorescence intensities over the range 830−870
nm under which one-photon hot band absorption phenomena
were absent. All hybrids exhibit quite good TPA properties,
with two-photon absorption cross section values ranging
between 159 and 527 GM. Two-photon absorption appears
Zemplen
́
’s method47 in quantitative yields to afford compound
5. No anisotropic effect of the macrocycle could be observed on
1
the H NMR spectrum for the mannosyl hydrogens signals,
proving that the carbohydrate moieties are located close to the
equatorial plane of macrocyle. All final compounds were
1
isolated in their E configuration as observed by H NMR
(3JH−H′ ≈ 16 Hz).
Photophysical Properties. The photophysical properties
of compounds 1−5 are presented in Table 1. Absorption
spectra of compounds 1−3 recorded in DMSO (Figure 2) are
typical of π-conjugated porphyrins23 with an intense Soret band
between 435 and 448 nm. The zinc complexes 1 and 3 exhibit
two Q bands at 580−584 and 634 nm, the latter one being
more intense while the free-base analogue 2 shows three Q
bands (521, 580, 669 nm). Compound 2 shows an additional
band at 376 nm which corresponds to the absorption of the
triphenylamine group (TP band). TP absorption is also
observed at the same wavelength for 1 and 3 but only as a
sideband. Compounds 4 and 5 show similar features in DMF.
Excitation of compounds 1−3 in CH2Cl2 at 450 nm (Soret
band) led to fluorescence emission above 600 nm characteristic
of porphyrin with two vibrational bands (Figure 3). Excitation
of the triphenylamine part at 380 nm led to the apparition of an
additional broad emission centered at 480 nm which was
attributed to the fluorescence of the triphenylamine part.
Substituted triphenylamines are known for being strongly
fluorescent molecules,48 the weak emission of the triphenyl-
amine part in the hybrids might result from an internal energy
transfer from the triphenylamine to the porphyrin. This
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a
Scheme 4. Synthesis of Compound 5
a
Key: (i) BBr3, CH2Cl2, −20 °C, 20 min; (ii) 2-[2-(2-bromoethoxy)ethoxy]-O-2′,3′,4′,6′-tetraacetyl-α-D-mannose, Cs2CO3, DMF, rt, 24 h; (iii)
NBS, CHCl3, pyridine, 0 °C, 15 min; (iv) Zn(OAc)2, CHCl3, MeOH, reflux, 5 min; (v) TMSA, Pd(PPh3)2Cl2, CuI, THF, Et3N, rt, 4 h; (vi) TBAF,
CH2Cl2, 0 °C, 30 min then 7b, Pd2(dba)3, AsPh3, THF, Et3N, rt, 20 h; (vii) PyrCH2PO(OEt)2, NaH, THF, rt, 48 h; (viii) NaOMe/MeOH, THF, rt,
1 h.
slightly better for compound 1 than for compound 2, which
points at the role played by the zinc atom in the 2PA
mechanism. Benzimidazolyl moieties (compound 3) led to a
higher δmax value than pyridyl (compound 2), probably due to
higher 2PA properties of Bzim compounds compared to
pyridiniums as was previously reported.51 It is noteworthy that
the modification on the meso positions of the porphyrin did not
significantly impact the 2PA properties (compounds 1, 4, and
5).
Photobiological Properties. Phototoxicity studies were
conducted on two tumor cell lines: Y79 is a commercial line of
human retinoblastoma cells overexpressing α-mannose specific
lectins,31 while HT-29 (human colon tumor cells) do not. Only
the biologically optimized compounds 4 and 5 were tested.
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Table 1. Photophysical Parameters of Porphyrin Derivatives
a
1−5
λabs (nm) ε (mM−1 cm−1
)
λem (nm)
ΦF
ΦΔ
δmax (GM)
1
2
448
580
634
376
435
521
580
669
380
446
584
634
451
582
632
450
581
639
160.0
10.6
30.7
103.5
210.0
20.5
33.2
15.8
75.9
359.3
30.1
82.6
228.8
12.9
40.3
159.2
5.8
649
0.08
0.80
251
678
743
0.06
0.09
0.64
159
527
3
649
Figure 4. Emission spectra of compounds 1, 8b, and 12 at 1 μM in
CH2Cl2. Excitation at 380 nm.
4
5
643
708
0.03
0.02
0.74
0.84
317
225
These tests showed that none of the hybrids displayed any
toxicity (dark or light, Table 2) on any cell line. These results
643
708
Table 2. Dark Toxicity and Phototoxicity (with Light
Illumination: 5 J/cm2 and Excitation between 400 and 800
nm) of 4 and 5 in Y79 and HT-29 Cell Lines
29.8
a
λabs = maximun absorption peak wavelength in DMSO, ε = molar
extinction coefficient. λem = emission peak in DMF with excitation in
Soret band, ΦF = fluorescence emission quantum yield in DMSO vs
coumarin 153 in EtOH (ΦF = 0.38), ΦΔ = singlet oxygen quantum
yield in DMF vs rose bengal (ΦΔ = 0.47), δ: 2PA cross-section at 830
nm in DMSO.
dark toxicity IC50 (μM)
photocytotoxicity IC50 (μM)
compd
Y79
HT-29
Y79
HT-29
4
5
>7.5
>7.5
>7.5
>7.5
>7.5
>7.5
>7.5
>7.5
were surprising given the high ΦΔ values of both compounds.
Furthermore, they reveal that the active targeting of the
membrane receptors with carbohydrates is inefficient. Both
observations could be linked to a poor internalization of the
hybrids in the cells.
The uptake of compounds 4 and 5 in Y79 and HT-29 cells
was evaluated by flow cytometry. Two excitation wavelengths
were used at 488 nm (foot of the Soret band) and 635 nm (Q-
band), and fluorescence emission was monitored above 670 nm
in the former case and between 653 and 669 nm for the latter.
Although these spectral characteristics were not optimal for
PTP compounds, we observed that both hybrids led to partial
staining of the cells of both lines with weak fluorescence
intensity. The case of 4 on HT-29 showed the highest staining
(81−88%). The spectroscopic differences between the two
compounds (ε, ΦF) prevent further comparison.
Figure 2. Normalized absorption spectra of compounds 1−3 in
DMSO.
Figure 3. Normalized emission spectra of compounds 1−3 in CH2Cl2. Excitation at 450 nm (left) and 380 nm (right).
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plates (SIL G-200, 2 mm) were used for preparative thin-layer
chromatography. Yields refer to chromatographically and spectroscopi-
Table 3. Uptake of Compounds 4 and 5 in Y79 and HT-29
Cell Lines, Compound Detection with the FACSCalibur
Characteristics, Cell Percentage of 4 or 5 Positive Cells, and
MFI (Treated/Controls Cells) of Fluorescence Intensity
1
cally pure compounds. H and 13C NMR spectra were recorded on a
spectrometer (300 MHz) at ambient temperature using an internal
deuterium lock. Chemical shift values are given in ppm relative to
tetramethylsilane (TMS). Acidic impurities in CDCl3 were removed
by treatment with anhydrous K2CO3. Melting points were performed
on a system Kofler.
General Procedures. Procedure A (Horner−Wadsworth−
Emmons Reaction). Sodium hydride (60% dispersion in mineral
oil) was washed with cyclohexane and introduced in a previously dried
flask purged with argon. The phosphonate was added and the mixture
stirred for 10−15 min at room temperature. The aldehyde, dissolved in
dry THF, was then added to the phosphonate via syringe. After
completion, the mixture was partitioned between H2O and CH2Cl2.
The aqueous phase was extracted three times with CH2Cl2. The
organic phases were reunited, washed with water, and dried with
anhydrous magnesium sulfate. After filtration, the solvents were
evaporated, and the crude product was purified by chromatography to
afford the desired products.
Procedure B1 (Sonogashira Coupling with TMSA). The bromozinc
derivative was combined with trans-dichlorobis(triphenylphosphine-
palladium(II) and copper(I) iodide. The flask was purged with argon
for 10 min. The solids were dissolved in dry THF and dry
triethylamine. The solution was deoxygenated with argon for 5 min
and then frozen at 77 K (liquid nitrogen). Trimethylsilylacetylene
(TMSA) was added, and the frozen mixture was left under argon flow
for 1 h. The gas was then allowed to escape, and the crude solution
warmed to room temperature was stirred for 16 h under argon after
which the reaction was quenched with water. It was then extracted
with CH2Cl2 and washed three times with water and once with brine.
The organic phase was dried over anhydrous magnesium sulfate and
filtered, and the solvents were removed under reduced pressure. The
crude product purified on silica gel column.
The lack of phototoxicity of the hybrids can therefore be
explained by a poor internalization combined with inappro-
priate intracellular localization or no internalization linked with
membrane binding and stacking due to the hydrophobicity of
the triphenylamine part. We previously showed the importance
of hydrophobicity in the case of our dimers of conjugated
porphyrins.39
Procedure B2 (Trimethylsilyl Deprotection Followed by Sonoga-
shira Coupling). To a solution of the silylated derivative in dry THF
and/or dry CH2Cl2 was slowly added a solution of tetrabutylammo-
nium fluoride (TBAF) 1 M solution in THF. A spatula of CaCl2 was
added to quench the reaction after 30 min at 20 °C. The organic layer
was concentrated under vacuum, the residue was dissolved in CH2Cl2,
and the solution was washed with water, dried over anhydrous
magnesium sulfate, filtered, and then concentrated under vacuum. The
generated product was combined with copper(I) iodide and
dichlorobis(triphenylphosphine)palladium(II) in anhydrous THF
and dry Et3N. The reaction mixture was stirred at room temperature
overnight. The solution diluted in CH2Cl2 was washed with water, the
aqueous layer was extracted with CH2Cl2, and the combined organic
layers were washed with water, dried over anhydrous magnesium
sulfate, filtered, and concentrated under vacuum. The products were
purified by silica gel chromatography.
Procedure C (Heck Coupling). The trimethylsilyl-protected
compound was dissolved in CH2Cl2, and TBAF (1 M in THF) was
added. The solution was stirred at room temperature for 15 min
followed by addition of anhydrous CaCl2. The mixture was stirred for
10 min and filtered, and the solvents were evaporated. The crude
product was combined with the adequate iodo or bromo derivative,
tris(dibenzylideneacetone)dipalladium(0), and triphenylarsine in
anhydrous THF and dry Et3N under argon. Depending on the
reactants, the reaction mixture was allowed to sit at room temperature
or heated to reflux under argon. The solvents were evaporated under
reduced pressure after which the crude product was taken up in
CH2Cl2, washed with water, dried over anhydrous magnesium sulfate,
and filtered. The solvents were removed under vacuum, and the crude
product was purified by chromatography and/or crystallization.
Procedure D (Deprotection of the Alkoxy Groups). The alkoxy-
protected porphyrin was dissolved in dry CH2Cl2 under argon and
cooled to −30 °C upon which boron tribromide BBr3 was added
cautiously. The temperature was maintained between −30 and −20 °C
for 20 min. The mixture was slowly poured into a water−ice bath after
which a solution of aqueous ammonia was added until the color
CONCLUSION
■
During this study, five potential 2PA photosensitizers were
synthesized. Their scaffold relies on the electronic conjugation
of a porphyrin core and a triphenylamine part via an alkyne
bond. Variations of the meso groups (Ph, Ph-TEGOMe and Ph-
DEG-mannose) or triphenylamine moieties (Pyr, Bzim) as well
as zinc internalization allowed discussion over the spectroscopic
properties of the final compounds.
The hybrids displayed high singlet oxygen production
quantum yields as well as good 2PA cross sections which
encouraged us to test them in 1PA phototoxicity assays.
Unfortunately, the compounds optimized for biological media
(4, 5) proved to be inactive against usual cancer line HT-29 as
well as retinoblastoma cells Y79. These observations were
attributed to poor internalization and stacking due to the
hydrophobicity of the compounds.
Further optimization has been undertaken so as to remedy to
this problem with the functionalization of the TP part with
hydrophilic groups.
EXPERIMENTAL SECTION
■
Synthesis. All solvents used were reagent grade. The following
reagents have been abbreviated: dimethylformamide (DMF), dimethyl
sulfoxide (DMSO), fetal calf serum (FCS), 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate buffer saline
(PBS), and Dulbecco’s modified Eagle’s medium (DMEM). 2-[2-(2-
Methoxyethoy)ethoxy]ethanol tosylate 14 (CAS [77544-60-6]) was
used without purification. Dry MeOH was kept over 3 Å sieves, and
dichloromethane was distilled from calcium hydride and kept over 4 Å
sieves. DMF was distilled under slow argon flow and kept over 4 Å
sieves. Column chromatography was performed with the indicated
solvents using silica gel 60 (particle size 0.035−0.070 mm). Precoated
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1
change from green to purple was complete. The mixture was extracted
with AcOEt, washed three times with water, dried over anhydrous
magnesium sulfate, and filtered. Solvents were removed under reduced
pressure and the solid residues dried under vacuum. Crystallization in
acetone/n-heptane allowed the isolation of the deprotected com-
pound.
Procedure E (Bromination of the Meso Position of Porphyrins).
The porphyrin was dissolved in CHCl3 and pyridine, and the resulting
solution was cooled to 0 °C. N-Bromosuccinimide (NBS) was added
and the mixture stirred for 15 min while slowly being warmed to room
temperature. Acetone was then introduced to quench the remaining
NBS. The solvents were removed under reduced pressure. The
remaining solid was then washed with water and dried.
Procedure F (Metalation). The porphyrin was dissolved in CHCl3
or acetone. A solution of zinc(II) acetate in methanol was added, and
the mixture was heated at reflux for 5 min. After the mixture was
cooled to room temperature and solvents were removed under
reduced pressure, the solid residue was taken up in CH2Cl2 and
washed with water. The organic phase was dried over anhydrous
magnesium sulfate and filtered, and the solvents were removed under
reduced pressure. The expected compound was then dried under
vacuum.
Procedure G (Williamson’s Reaction). A mixture of hydroxylated
porphyrins, bromo-polyethylene glycol derivatives, and cesium
carbonate in dry DMF was stirred under argon at room temperature
during 24 h. The mixture was concentrated under vacuum and taken
up with a mixture of water and ethyl acetate (2/1, v/v), and the
organic layer was separated. The aqueous layer was extracted with
ethyl acetate. The combined organic extracts were washed with water
(2×), dried over magnesium sulfate, and filtered, and the solvent was
evaporated under vacuum. The product was purified by three
crystallizations from CH2Cl2/n-heptane and then purified by
preparative chromatography.
powder (θf = 120 °C, 91% yield): H NMR (CDCl3, 300 MHz) 9.90
(s, 2H), 7.79 (d, J = 8.5 Hz, 4H), 7.46 (d, J = 8.4 Hz, 2H), 7.19 (d, J =
8.5 Hz, 4H), 7.09 (d, J = 8.4 Hz, 2H), 0.25 (s, 9H); 13C NMR (CDCl3,
75.3 MHz) 190.5, 151.6, 145.6, 133.7, 131.8, 131.4, 126.1, 123.4,
120.5, 104.2, 95.3, 0.0; MS (ESI+) m/z [M + Na]+ calcd for
C25H23NNaO2Si 420.0, found 420.0; HRMS (MALDI-TOF) (m/z)
[M]+ calcd for C25H23NO2Si 397.1498, found 397.1506.
4,4′-(4-Ethynylphenylazanediyl)dibenzaldehyde (TPY-2CHO, 10).
Compound 9 (208 mg, 0.52 mmol) in CH2Cl2 (40 mL) was
deprotected at 0 °C with 0.62 mL of TBAF (1 M in THF). The
reaction was stirred at 0 °C for 30 min. The solution was filtered over a
short pad of silica and eluted with a mixture CH2Cl2/i-PrOH (9/1, v/
v). The solvents removed under vacuum to afford 200 mg (quant) of
1
the title compound as a dark yellow solid (θf = 99 °C): H NMR
(CDCl3, 300 MHz) 9.84 (s, 2H), 7.72 (d, J = 8.4 Hz, 4H), 7.41 (d, J =
8.3 Hz, 2H), 7.12 (d, J = 8.4 Hz, 4H), 7.04 (d, J = 8.3 Hz, 2H), 3.05 (s,
1H); 13C NMR (CDCl3, 75.3 MHz) 188.9, 150.0, 144.4, 132.3, 130.2,
129.8, 124.5, 121.8, 117.8, 81.3, 76.5; MS (ESI+) m/z [M + H]+ calcd
for C22H16NO2 326.1, found 326.2; HRMS (MALDI-TOF) (m/z)
[M]+ calcd for C22H15NO2 325.1103, found 325.1091.
PTPM-2CHO (12). According to procedure C, a flask was charged
with TPY-2CHO (201 mg, 0.47 mmol, 2 equiv), TPPMBr (150 mg,
0.24 mmol, 1 equiv), Pd2(dba)3 (17 mg, 0.07 mmol, 0,3 equiv), and
AsPh3 (110 mg, 0.36 mmol, 1.5 equiv), and purged for 10 min with
argon. The solids were dissolved in dry THF (20 mL), and the
solution was deoxygenated for 20 min before the addition of dry Et3N
(4 mL). The reaction was stirred for 20 h at room temperature. The
solvents were removed under reduced pressure, and the residue was
purified by silica gel column chromatography (CHCl3/Et2O, 98/2, v/
v) to afford 250 mg (84%) of PTPM-2CHO as a dark blue-green
powder: UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 444 (419.0), 569
1
(26.7), 618 (43.5); H NMR (CDCl3, 300 MHz) 9.93 (s, 2H), 9.83
(d, J = 4.6 Hz, 2H), 8.95 (d, J = 4.6 Hz, 2H), 8.80 (s, 4H), 8.24 (m,
6H), 8.10 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.6 Hz, 4H), 7.79 (m, 9H),
7.40 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 4H); 13C NMR (CDCl3,
75.3 MHz) 187.5, 150.3, 149.7, 148.6−147.8, 144.0, 141.4−141.3,
132.5−132.4, 131.1, 130.5, 130.4, 129.7−129.4, 129.0, 128.3, 125.5,
124.5−124.4, 121.5, 120.7−119.7, 97.0, 93.1, 92.0; MS (MALDI-
TOF) m/z [M]+ calcd for C60H37N5O2Zn 923.22, found 923.21;
HRMS (MALDI-TOF) (m/z) [M]+ calcd for C60H37N5O2Zn
923.2239, found 923.2273.
PTP-2CHO (13). Compound 12 (250 mg, 0.27 mmol) was dissolved
in CHCl3 (20 mL) and introduced in a separatory funnel. TFA (1 mL)
was added, and the funnel was shaken for a few minutes. The mixture
was washed with water until pH 7 was reached. The organic phase was
dried over magnesium sulfate and filtered, and the solvents were
removed under reduced pressure. Purification was achieved by
preparative TLC (AcOEt/cyclohexane, 30/70, v/v) to afford the
desired compound as a blue solid (147 mg, 63%): UV−vis in CH2Cl2
λmax nm (ε mM−1 cm−1): 437 (385.3), 532 (24.9), 581 (49.5), 615
4-Bromo-N,N-bis(4-((E)-2-(pyridin-4-yl)vinyl)phenyl)aniline (TPBr-
2Py, 8a). Compound 8a was obtained following procedure A with
diethyl[(pyridin-4-yl)methyl]phosphonate (900 mg, 3.9 mmol, 3
equiv) in dry THF (15 mL), NaH (110 mg, 4.6 mmol, 3.5 equiv),
and TPBr-2CHO (500 mg, 1.32 mmol, 1 equiv) in dry THF (15 mL).
The residue was purified over a silica gel column eluted with CH2Cl2/
i-PrOH (95/5, v/v): 322 mg of yellow solid was isolated (θf = 176 °C,
1
46% yield); H NMR (CDCl3, 300 MHz) 8.55 (d, J = 5.1 Hz, 4H),
7.44 (d, J = 8.6 Hz, 4H), 7.40 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 5.8 Hz,
4H), 7.26 (d, J = 15.8 Hz, 2H), 7.09 (d, J = 8.6 Hz, 4H), 7.02 (d, J =
8.8 Hz, 2H), 6.92 (d, J = 16.3 Hz, 2H); 13C NMR (CDCl3, 75.3 MHz)
150.1, 147.3, 146.0, 144.8, 132.5, 132.3, 131.2, 128.2, 126.4, 124.7,
124.0, 120.7, 116.4; MS (ESI+) m/z [M + H]+ calcd for C32H25BrN3
530.1, found 530.1; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C32H24BrN3 529.1154, found 529.1148.
4-Iodo-N,N-bis(4-((E)-2-(pyridin-4-yl)vinyl)phenyl)aniline (TPI-
2Py, 8b). Following procedure A, the title product was obtained
from TPI-2CHO (500 mg, 1.32 mmol, 1 equiv), diethyl[(pyridin-4-
yl)methyl]phosphonate (907 mg, 3.96 mmol, 3 equiv), and NaH (110
mg, 4.62 mmol, 3.5 equiv) in dry THF (30 mL). The crude product
was purified by silica gel chromatography eluted by a mixture of
CH2Cl2/i-PrOH (95/5, v/v) and yielded 488 mg of the desired
1
(15.8), 670 (23.9); H NMR (CDCl3, 300 MHz) 9.95 (s, 2H), 9.72
(d, J = 4.8 Hz, 2H), 8.92 (d, J = 4.7 Hz, 2H), 8.76 (s, 4H), 8.19 (m,
6H), 8.02 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.6 Hz, 4H), 7.78 (m, J =
6.2 Hz, 9H), 7.33 (m, J = 8.4 Hz, 6H), −2.31 (s, 2H); 13C NMR
(CDCl3, 75.3 MHz) 190.7, 141.7, 134.5, 133.3, 131.5, 127.9, 126.8,
126.5, 123.5, 121.2; MS (MALDI-TOF) m/z [M]+ calcd for
C60H39N5O2 861.31, found 861.36.
1
compound as an orange powder (θf = 117 °C, 64% yield): H NMR
(CDCl3, 300 MHz) 8.56 (s, 4H), 7.58 (d, J = 8.7 Hz, 2H), 7.44 (d, J =
8.5 Hz, 4H), 7.35 (d, J = 4.7 Hz, 4H), 7.26 (d, J = 16.3 Hz, 2H), 7.09
(d, J = 8.5 Hz, 4H), 6.95−6.88 (m, 4H); 13C NMR (CDCl3, 75.3
MHz) 150.2, 147.2, 146.7, 144.7, 138.5, 132.3, 131.3, 128.2, 126.6,
124.8, 124.1, 120.7, 86.8; MS (ESI+) m/z [M + H]+ calcd for
C32H25IN3 578.1, found 578.0.
4,4′-(4-((Trimethylsilyl)ethynyl)phenylazanediyl)dibenzaldehyde
(TPYSi-2CHO, 9). Following procedure B1, TPI-2CHO (630 mg, 1.47
mmol, 1 equiv) was combined with Pd2(PPh3)2Cl2 (105 mg, 0.15
mmol, 0.1 equiv) and CuI (28 mg, 0.15 mmol, 0.1 equiv) in dry THF
(10 mL). TMSA (1.25 mL, 8.8 mmol, 6.0 equiv) and dry Et3N (10
mL) were added via syringe. The reaction was purified by flash
chromatography over silica (eluted with ethyl acetate/cyclohexane
30:70, v/v). The title product (533 mg) was obtained as a yellow
5-H-10,15,20-Tri-p-phenol Porphyrin (TPP(OH)3, 15). Following
procedure D, TPP(OiPr2OH)3 (800 mg, 1.19 mmol, 1 equiv) was
44
dissolved in dry CH2Cl2 (100 mL), then BBr3 (2.26 mL, 23.8 mmol,
20 equiv) was added. TPP(OH)3 was precipitated from AcOEt with n-
heptane to afford 595 mg of purple powder (yield 85%): UV−vis in
acetone λmax nm (ε mM−1 cm−1) 412 (420), 509 (15.3), 545 (7.8),
586 (4.5), 642 (3.3); 1H NMR (acetone-d6, 300 MHz) 10.17 (s, 1H),
9.30 (d, J = 4.6 Hz, 2H), 8.98 (d, J = 4.6 Hz, 2H), 8.3 (m, 4H), 8.03 (t,
J = 8.6 Hz, 6H), 7.28 (d, J = 8.4 Hz, 6H), −2.98 (s, 2H); 13C NMR
(acetone-d6, 75.3 MHz) 170.2, 170.1, 158.7, 135.7−135.2, 134.4,
131.45, 120.3, 119.3, 113.1, 112.7, 97.7 (C-20); MS (MALDI-TOF)
m/z [M]+ calcd for C38H26N4O3 586.20, found 586.21; HRMS
1413
dx.doi.org/10.1021/jo402658h | J. Org. Chem. 2014, 79, 1406−1417

The Journal of Organic Chemistry
Article
(MALDI-TOF) (m/z) [M]+ calcd for C38H26N4O3 586.2005, found
586.2005.
Hz, 4H), 8.86 (s, 4H), 8.07−8.01 (m, 6H), 7.95 (d, J = 8.5 Hz, 2H),
7.66 (d, J = 8.7 Hz, 4H), 7.14 (m, 6H), 7.05 (d, J = 8.7 Hz, 4H), 4.08
(m, 6H), 3.70 (m, 6H), 3.51 (m, 6H), 3.42−3.31 (m, 12H), 3.23 (m,
6H), 3.09 (s, 9H); 13C NMR (CDCl3, 75.3 MHz) 190.5, 158.3, 152.1,
151.5, 150.9, 150.3, 150.0, 135.4, 135.2, 133.1, 131.5, 131.3, 126.5,
123.1, 121.7, 112.7, 99.0, 95.1, 94.2, 71.6−67.4, 58.8; MS (MALDI-
TOF) m/z [M]+ calcd for C81H79N5O14Zn 1409.49, found 1409.50;
HRMS (MALDI-TOF) (m/z) [M]+ calcd for C81H79N5O14Zn
1409.4915, found 1409.4908.
5,10,15-Tri[(p-2′,3′,4′,5′,6′-tetracacetyl-α-D-mannosyloxy-2-
ethoxy)-2-ethyloxyphenyl]porphyrin (TPP(DEGManOAc)3, 21).39 5-
Allyl-10,15,20-tri[(p-2′,3′,4′,5′,6′-tetracetyl-α-D-mannosyloxy-2-
ethoxy)-2-ethyloxyphenyl]porphyrin Zinc Complex
(TPPM (DEGManOAc)3YSi, 22).39 5-Allyl-10,15,20-tri(p-
hydroxyphenyl)porphyrin Zinc Complex (TPPM(OH)3YSi, 23).
Following procedures E, F, and B1, starting from 15, the expected
compound 23 was isolated after purification by preparative TLC
eluted with CH2Cl2/EtOH (95/5, v/v) as a dark blue solid (32 mg,
59%): UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 431 (122.3), 562
(5.7), 609 (4.4); 1H NMR (acetone-d6, 300 MHz) 9.62 (d, J = 4.6 Hz,
2H), 8.90 (d, J = 4.6 Hz, 2H), 8.81−8.77 (m, 4H), 7.92 (m, 6H), 7.19
(m, 8.3 Hz, 6H), 0.60 (s, 9H); 13C NMR (acetone-d6, 75.3 MHz)
158.1, 153.1−151.0, 136.3, 136.2−132.2, 122.6, 114.4, 55.4, 0.5; MS
(MALDI-TOF) m/z [M]+ calcd for C43H32N4O3SiZn 744.15, found
744.11; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C43H32N4O3SiZn 744.1535, found 744.1547.
PTPM(OH)3-2CHO (24). The title compound was obtained following
procedure C starting from 23 (265 mg, 0.36 mmol, 1 equiv) and
TBAF (0.47 mL at 1 M in THF, 0.47 mmol, 1.3 equiv) in dry CH2Cl2
(5 mL) and dry THF (25 mL) first followed by 7b (303 mg, 0.71
mmol, 2 equiv) in the presence of Pd2dba3 (25 mg, 0.11 mmol, 0.3
equiv) and AsPh3 (165 mg, 0.54 mmol, 1.5 equiv) in dry THF (20
mL) and dry Et3N (10 mL). Purification over silica gel eluted with
CH2Cl2/EtOH (95/5, v/v) afforded the desired compound with a
69% yield (243 mg) as a dark blue powder: UV−vis in THF λmax nm
(ε mM−1 cm−1) 446 (173.7), 564 (7.3), 630 (17.3); 1H NMR
(acetone-d6, 300 MHz) 9.69 (s, 2H), 9.65 (d, J = 4.6 Hz, 2H), 8.84 (d,
J = 4.6 Hz, 2H), 8.70 (s, 4H), 7.90 (d, J = 8.4 Hz, 2H), 7.84 (t, J = 7.9
Hz, 6H), 7.60 (d, J = 8.5 Hz, 4H), 7.10 (m, 11H), 7.01 (d, J = 8.5 Hz,
4H); 13C NMR (acetone-d6, 75.3 MHz) 191.2, 158.1, 152.9, 152.5,
152.4, 151.8, 151.1, 150.9, 146.5, 138.2, 136.4, 134.9, 132.9, 132.4,
132.0, 129.1, 127.5, 126.1, 124.2, 114.5, 99.3, 95.9, 94.8; MS (MALDI-
TOF) m/z [M]+ calcd for C60H37N5O5Zn 971.21, found 971.20;
HRMS (MALDI-TOF) (m/z) [M]+ calcd for C60H37N5O5Zn
971.2086, found 971.2088.
5-Bromo-10,15,20-tri-p-phenol Porphyrin (TPP(OH)3Br, 16). The
title compound was obtained through procedure E from TPP(OH)3
(183 mg, 0.23 mmol, 1 equiv). TPP(OH)3Br was isolated as a dark
blue powder (149 mg, 97%) and used without purification: UV−vis in
CH2Cl2 λmax nm (ε mM−1 cm−1) 424 (218.7), 523 (9.6), 560 (7.1),
600 (3.2), 658 (3.8); 1H NMR (acetone-d6, 300 MHz) 9.60 (d, J = 4.8
Hz, 2H), 8.94 (d, J = 4.5 Hz, 2H), 8.89 (s, 4H), 8.03 (d, J = 8.4 Hz,
6H), 7.33−7.28 (m, 6H), 2.96 (broad s, 3H), −2.74 (s, 2H); 13C
NMR (acetone-d6, 75.3 MHz) 158.6, 136.6, 136.4, 133.7, 133.5, 122.4,
121.9, 114.8, 102.2; MS (MALDI-TOF) m/z [M]+ calcd for
C38H25BrN4O3 664.11, found 664.11; HRMS (MALDI-TOF) (m/z)
[M]+ calcd for C38H25BrN4O3 664.1110, found 664.1109.
5-Bromo-10,15,20-tri-p-phenol Porphyrin Zinc Complex
(TPPM(OH)3Br, 17). Procedure F afforded the title compound starting
from TPP(OH)3Br (130 mg, 195 mmol, 1 equiv) and zinc(II) acetate
(179 mg, 977 mmol, 5.0 equiv) in 70 mL of acetone and 30 mL of
MeOH. The solvents were evaporated, and 17 was isolated as a purple
powder (120 mg, 84% yield): UV−vis in CH2Cl2 λmax nm (ε mM−1
1
cm−1) 430 (111.8), 566 (4.4), 640 (7.2); H NMR (acetone-d6, 300
MHz) 9.70 (d, J = 4.7 Hz, 2H), 9.00 (d, J = 4.7 Hz, 2H), 8.91 (s, 4H),
8.05−8.00 (m, 6H), 7.29 (dd, J1 = 11.4 Hz, J2 = 4.9 Hz, 6H), 2.77 (s,
3H); 13C NMR (acetone-d6, 75.3 MHz) 158.1, 151.8−150.1, 136.4,
136.3, 135.0, 134.9, 133.8, 132.9, 132.8, 122.7, 122.3, 114.4, 103.6,
100.9; MS (MALDI-TOF) m/z [M]+ calcd for C38H23BrN4O3Zn
726.02, found 726.02; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C38H23BrN4O3Zn 726.0245, found 726.0274.
5-Bromo-10,15,20-tri(p-2-(2-(2-methoxyethoy)ethoxy)-
ethanoxyphenylporphyrin Zinc Complex (TPPM(OTEG)3Br, 18).
According to procedure G, TPPM(OH)3Br (660 mg, 0.87 mmol, 1
equiv), TEGOTs (4.15 g, 13.05 mmol, 15 equiv), and Cs2CO3 (12.76
g, 40 mmol, 45 equiv) reacted in anhydrous DMF (100 mL) to afford
18 as a purple solid after recrystallization from CH2Cl2/n-heptane
(913 mg, 88%): UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 429
(207.0), 564 (7.5), 605 (5.0); 1H NMR (CDCl3, 300 MHz) 9.74 (d, J
= 4.6 Hz, 2H), 8.98 (d, J = 4.6 Hz, 2H), 8.88 (s, 4H), 8.07−8.04 (m,
6H), 7.20−7.17 (m, 6H), 4.19 (m, 6H), 3.81 (m, 6H), 3.62−3.61 (m,
6H), 3.51−3.44 (m, 12H), 3.35 (m, 6H), 1.43 (s, 9H); 13C NMR
(CDCl3, 75.3 MHz) 158.4, 150.9, 149.6, 135.4, 132.2, 121.3, 112.7,
71.7−67.5, 58.9; MS (MALDI-TOF) m/z [M]+ calcd for
C59H65BrN4O12Zn 1164.31, found 1164.31; HRMS (MALDI-TOF)
(m/z) [M]+ calcd for C59H65BrN4O12Zn 1164.3074, found 1164.3014.
5-Allyl-10,15,20-tri(p-2-(2-(2-methoxyethoy)ethoxy)-
ethanoxyphenylporphyrin Zinc Complex. TPPM(OTEG)3YSi (19).
Following procedure B1, TPPM(OTEG)3Br (0.80 g, 0.69 mmol, 1
equiv) was combined with TMSA (390 mg, 4.1 mmol, 6 equiv),
Pd(PPh3)2Cl2 (47 mg, 69 mmol, 0.1 equiv), and CuI (12 mg, 69
mmol, 0.1 equiv) in dry THF (15 mL) and dry Et3N (2.5 mL). The
title compound was obtained after purification over silica gel
chromatography (CH2Cl2/MeOH, 99/1, v/v) as a blue powder
(560 mg, 69% yield): UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 435
(259.8), 572 (9.2), 619 (11.6); 1H NMR (CDCl3, 300 MHz) 9.72 (d, J
= 4.6 Hz, 2H), 8.96 (d, J = 4.6 Hz, 2H), 8.84 (m, 4H), 8.03 (m, 6H),
7.09 (m, 6H), 4.01 (m, 6H), 3.62 (m, 6H), 3.42 (m, 6H), 3.31 (m,
6H), 3.23 (m, 6H), 3.14 (m, 6H), 2.99 (s, 9H), 0.62 (s, 9H); 13C
NMR (CDCl3, 75.3 MHz) 157.8, 152.0, 150.3, 149.6, 134.9, 135.2,
132.3, 131.6, 130.2, 121.0, 112.2, 98.5, 95.6, 93.7, 71.0−69.0, 58.2, 0.0;
MS (MALDI-TOF) m/z [M]+ calcd for C64H74N4O12SiZn 1182.44,
found 1182.44.
PTPM(DEG(Man(OAc)4)3-2CHO (25). The title compound was
obtained through procedure G from 24 (55 mg, 0.06 mmol, 1
equiv) with Cs2CO3 (159 mg, 2.70 mmol, 45 equiv) and [(2-
bromoethoxy)-O-ethoxy]-O-2′,3′,4′,6′-tetraacetyl-α-D-mannose (427
mg, 0.90 mmol, 15 equiv). Compound 25 was isolated after
purification over silica gel eluted with cyclohexane/AcOEt (7/3, v/
v) as a dark blue powder (19 mg, 70%): UV−vis in CH2Cl2 λmax nm (ε
1
mM−1 cm−1) 447 (136.5), 576 (6.8), 628 (17.4); H NMR (CDCl3,
300 MHz) 9.80 (s, 2H), 9.01 (s, 4H), 8.87 (s, 4H), 8.08 (s, 6H), 7.97
(d, J = 7.9 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.30 (m, 8H), 7.10 (d, J
= 7.4 Hz, 2H), 5.42−5.33 (m, 9H), 4.96 (s, 3H), 4.40−4.31 (m, 9H),
4.17 (s, 6H), 4.02 (s, 6H), 3.85 (s, 12H), 2.16 (s, 9H), 2.11 (s, 9H),
2.03 (s, 9H), 1.99 (s, 9H); 13C NMR (CDCl3, 75.3 MHz) 170.7,
170.0, 158.5, 152.8, 150.9, 135.5, 133.0−129.7, 112.8, 97.8, 70.4, 70.0,
69.6, 69.2, 68.5, 67.5, 66.2, 62.5, 20.9, 20.7; MS (MALDI-TOF) m/z
[M + H]+ calcd for C114H116N5O38Zn 2227.52, found 2227.65; HRMS
(MALDI-TOF) (m/z) [M]+ calcd for C114H115N5O38Zn 2226.6545,
found 2226.6681.
5-Trimethylsilyl-10,15,20-tri(p-isopropyloxyphenyl)porphyrin
Zinc Complex (TPPM(OiPr)3YSi, 26). Following procedure B1,
TPPM(OiPr)3Br (691 mg, 0.81 mmol, 1 equiv) reacted with TMSA
(685 mL, 4.85 mmol, 6 equiv) in the presence of Pd(PPh3)2Cl2 (57
mg, 0.08 mmol, 0.1 equiv) and CuI (15 mg, 0.08 mmol, 0.1 equiv) in
dry THF (20 mL) and dry Et3N (1 mL). The desired compound was
isolated as a dark blue solid after purification over silica gel column
PTPM(OTEG)3-2CHO (20). Following procedure C, 19 (550 mg, 0.46
mmol, 1 equiv) was deprotected with TBAF (506 μL at 1 M in THF,
0.56 mmol, 1.2 equiv) in THF (40 mL) and CH2Cl2 (8 mL) and was
then reacted with TPI-2CHO (370 mg, 0.87 mmol, 1.9 equiv),
Pd2dba3 (30 mg, 0.13 mmol, 0.3 equiv), and AsPh3 (200 mg, 0.65
mmol, 1.5 equiv) in dry THF (20 mL) and dry Et3N (20 mL). Silica
gel chromatography eluted with CH2Cl2/EtOH (99/1 to 95/5, v/v)
afforded 650 mg of 20 (99% yield) as a dark blue powder: UV−vis in
CH2Cl2 λmax nm (ε mM−1 cm−1) 377 (20.0), 450 (203.4), 580 (8.2),
634 (22.3); 1H NMR (CDCl3, 300 MHz) 9.77 (s, 4H), 8.99 (d, J = 4.6
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eluted with CH2Cl2 (576 mg, 82%): UV−vis in CH2Cl2 λmax nm (ε
mM−1 cm−1): 433 (436.9), 567 (16.3), 612 (14.4); 1H NMR (CDCl3,
300 MHz) 9.71 (d, J = 4.4 Hz, 2H), 9.00 (d, J = 4.4 Hz, 2H), 8.88 (s,
4H), 8.06 (d, J = 8.0 Hz, 6H), 7.25 (m, 6H), 4.83 (sept, J = 5.7 Hz,
3H), 1.54 (d, J = 5.5 Hz, 18H), 0.61 (s, 9H); 13C NMR (CDCl3, 75.3
MHz) 158.0, 153.0−150.6, 136.0, 135.9, 135.4−135.3, 133.4−132.1,
131.2, 123.3, 122.2, 114.3−114.2, 108.2, 101.5, 70.6, 22.8, 0.9; MS
(MALDI-TOF) m/z [M]+ calcd for C52H50N4O3SiZn 870.29, found
870.26; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C52H50N4O3SiZn 870.2944, found 870.2916.
in DMF λmax nm (ε mM−1 cm−1) 450 (79.6), 581 (4.6), 638 (16.1);
1H NMR (CDCl3, 300 MHz) 9.59 (d, J = 4.3 Hz, 2H), 8.81 (d, J = 3.7
Hz, 2H), 8.71 (s, 4H), 7.98 (d, J = 7.4 Hz, 6H), 7.75 (d, J = 7.9 Hz,
2H), 7.25−7.01 (m, 13H), 6.91 (d, J = 7.5 Hz, 4H), 6.80−6.60 (m,
2H), 6.33 (m, 6H), 5.39−5.20 (m, 4.6 Hz, 9H), 4.90 (s, 3H), 4.28−
3.78 (4m, 36H), 2.09 (s, 9H), 2.05 (s, 9H), 1.95 (s, 9H), 1.91 (s, 9H);
13C NMR (CDCl3, 75.3 MHz) 169.7, 169.1, 168.9, 168.8, 157.2, 151.0,
149.7, 149.1, 148.8, 146.3, 145.6, 145.2, 144.0, 134.8, 134.4, 132.0,
131.6, 130.8, 129.6, 127.1, 123.9, 122.9, 122.4, 121.3, 120.3, 118.9,
111.5, 97.5, 96.8, 94.3, 93.0, 69.3, 69.0, 68.8, 68.6, 68.4, 68.1, 67.5,
66.9, 66.6, 66.5, 65.2, 62.6, 61.5, 20.0, 19.9, 19.8, 19.7, 19.7; MS
(MALDI-TOF) m/z [M + H]+ calcd for C126H126N7O36Zn 2377.75,
found 2377.70; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C126H125N7O36Zn 2376.7491, found 2376.7469.
PTPM(OiPr)3-2CHO (27). Following procedure H, 26 (370 mg, 0.42
mmol, 1 equiv) was put to reaction first with TBAF (1 M in THF) in
dry CH2Cl2 (7 mL) and dry THF (36 mL). The crude product was
combined with TPBr-2CHO (177 mg, 0.47 mmol, 1.1 equiv), Pd2dba3
(30 mg, 0.13 mmol, 0.3 equiv), and AsPh3 (193 mg, 0.63 mmol, 1.5
equiv) in dry THF (53 mL) and dry Et3N (11 mL). The crude was
purified twice over a silica gel column eluted with CH2Cl2/EtOH
(100/1 to 98/2, v/v) to afford the desired compound as a dark blue
solid (88 mg, 19%): UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 446
PTPM-2Py (1). Compound 1 was synthesized according to
procedure F from 12 (50 mg, 54 μmol, 1 equiv), PyrCH2PO(OEt)2
(37 mg, 162 μmol, 3 equiv), and NaH (8 mg, 189 μmol, 3.5 equiv) in
dry THF (2 mL). The crude product was purified by preparative TLC
(CH2Cl2/MeOH, 95/5, v/v) to afford 35 mg (64%) of the title
compound as a dark blue powder: UV−vis in DMSO λmax nm (ε
1
(231.1), 522 (3.2), 572 (12.1), 624 (25.9); H NMR (CDCl3, 300
MHz) 9.66 (d, J = 4.6 Hz, 2H), 9.56 (s, 2H), 8.98 (d, J = 4.6 Hz, 2H),
8.91 (d, J = 0.9 Hz, 4H), 8.04 (d, J = 8.4 Hz, 6H), 7.92 (d, J = 8.4 Hz,
2H), 7.60 (d, J = 8.6 Hz, 4H), 7.24 (d, J = 10.2 Hz, 6H), 7.15 (d, J =
8.5 Hz, 2H), 7.07 (d, J = 8.5 Hz, 4H), 4.91−4.69 (m, 3H,) 1.55 (d, J =
6.0 Hz, 18H); 13C NMR (CDCl3, 75.3 MHz) 190.4, 157.6, 152.0,
151.5, 150.9, 150.3, 150.0, 145.1, 135.6, 134.7, 134.6, 133.2, 132.9,
132.3, 131.9, 131.4, 131.3, 130.3, 126.4, 123.1, 121.9, 121.7, 113.9,
99.1, 95.2, 93.8, 70.1, 22.3; MS (MALDI-TOF) m/z [M]+ calcd for
C69H55N5O5Zn 1097.35, found 1097.36; HRMS (MALDI-TOF) (m/
z) [M]+ calcd for C69H55N5O5Zn 1097.3495 found 1097.3524.
PTPM(OiPr)3-2Py (28). Compound 27 (86 mg, 0.078 mmol, 1 equiv)
was converted to the title compound according to procedure A with
PyrCH2PO(OEt)2 (72 mg, 0.313 mmol, 4.0 equiv) and NaH (8 mg,
0.351 mmol, 4.5 equiv). Purification over a silica gel column eluted
with CH2Cl2/EtOH (99/1 to 95/5, v/v with a very weak gradient)
afforded the desired compound 28 as a dark blue solid (20 mg, 0.016
mmol, 21%): UV−vis in THF λmax nm (ε mM−1 cm−1) 430 (110.3),
521 (3.0), 592 (6.4), 681 (2.8); 1H NMR (CDCl3, 300 MHz) 9.66 (d,
J = 4.2 Hz, 2H), 8.91 (d, J = 5.0 Hz, 2H), 8.80 (s, 4H), 8.03 (d, J = 8.4
Hz, 6H), 7.81 (d, J = 9.1 Hz, 2H), 7.19 (m, 10H), 7.14−7.07 (m, 6H),
6.93 (d, J = 9.0 Hz, 4H), 6.73 (d, J = 10.2 Hz, 2H), 6.31 (m, 6H), 4.79
(m, 3H), 1.51 (d, J = 5.9 Hz, 18H); 13C NMR (CDCl3, 75.3 MHz)
157.4, 152.1, 150.8, 150.2, 149.9, 147.3, 146.7, 144.7, 135.6, 135.4,
132.8, 132.7, 130.7, 128.1, 124.7, 124.0, 121.5, 119.9, 113.7, 98.5, 95.3,
94.1, 70.1, 22.3; MS (MALDI-TOF) m/z [M]+ calcd for
C81H65N7O3Zn 1247.44, found 1247.43.
1
mM−1 cm−1) 448 (160.0), 580 (10.6), 634 (30.7); H NMR (CDCl3,
300 MHz) 9.61 (d, J = 4.5 Hz, 2H), 8.79 (d, J = 4.5 Hz, 2H), 8.67 (s,
4H), 8.06 (s, 6H), 7.73 (d, J = 8.3 Hz, 2H), 7.60 (m, 9H), 7.26 (d, J =
8.5 Hz, 2H), 7.08−7.01 (m, 4H), 6.86 (d, J = 8.4 Hz, 4H), 6.81 (d, J =
5.0 Hz, 2H), 6.73 (d, J = 16.3 Hz, 2H), 6.48 (s, 4H), 6.34 (d, J = 16.3
Hz, 2H), 6.09 (m, 2H); 13C NMR (CDCl3, 75.3 MHz) 151.1, 149.4,
148.8, 148.6, 147.3, 146.3, 146.2, 145.3, 143.7, 142.2, 142.1, 135.0,
133.5, 133.4, 131.7, 131.5, 130.9, 130.5, 129.7, 129.3, 127.0, 126.2,
125.3, 123.4, 123.1, 122.7, 122.4, 121.5, 120.6, 119.0, 118.4, 98.0, 94.4,
92.8; MS (MALDI-TOF) m/z [M]+ calcd for C72H47N7Zn 1073.32,
found 1073.31; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C72H47N7Zn 1073.3184, found 1073.3167.
PTP-2Py (2). Following procedure F, 13 (60 mg, 70 mmol, 1 equiv)
was reacted with PyrCH2PO(OEt)2 (48 mg, 210 μmol, 3 equiv) and
NaH (10 mg, 245 μmol, 3.5 equiv). The crude product was purified
over silica gel column chromatography (CH2Cl2/EtOH, 95/5, v/v) to
yield the title compound as a dark blue solid (30 mg, 42%): UV−vis in
DMSO λmax nm (ε mM−1 cm−1) 376 (103.5), 435 (210.0), 521 (20.5),
580 (33.2), 669 (15.8); 1H NMR (CDCl3, 300 MHz) 9.72 (d, J = 4.8
Hz, 2H), 8.90 (d, J = 4.7 Hz, 2H), 8.75 (s, 4H), 8.48 (d, J = 3.7 Hz,
4H), 8.20−8.15 (m, 6H), 7.89 (d, J = 8.6 Hz, 2H), 7.75−7.70 (m,
9H), 7.41 (d, J = 8.5 Hz, 4H), 7.32−7.08 (m, 12H), 6.85 (d, J = 16.3
Hz, 2H), −2.27 (s, 2H); 13C NMR (CDCl3, 75.3 MHz) 150.1, 147.2,
147.1, 144.8, 142.0−141.7, 134.5−134.4, 132.9, 132.4, 131.5, 128.3,
127.9, 126.9, 126.8, 124.8, 124.6, 124.1, 121.8−118.5, 120.7, 99.9, 96.9,
92.2; MS (MALDI-TOF) m/z [M]+ calcd for C72H49N7 1011.40,
found 1011.43; HRMS (MALDI-TOF) (m/z) [M]+ calcd for
C72H49N7 1011.4049, found 1011.4050. Anal. Calcd for C72H49N7·
14H2O: C, 68.39; H, 6.14; N, 7.75. Found: C, 68.04; H, 5.98; N, 7.13.
PTPM-2Bzim (3). Compound 12 (100 mg, 0.11 mmol, 1 equiv) was
dissolved in THF (5 mL) along with (1-methylbenzimidazol-2-
ylmethyl)triphenylphosphonium chloride (97 mg, 0.23 mmol, 2.2
equiv) and 1,8-diazabicyclo[5.4.0]undec-7-ene (83 μL, 0.55 mmol, 5
equiv). The mixture was stirred at room temperature in the dark for 15
h. The solvent was removed under reduced pressure and the residue
taken up in ethyl acetate, washed with water and then brine, dried over
magnesium sulfate, and filtered, and the solvents were removed. The
product was purified over silica gel column chromatography (CH2Cl2/
MeOH, 98/2, v/v) to afford 28 mg (22%) of the desired compound as
a dark blue powder: UV−vis in DMSO λmax nm (ε mM−1 cm−1) 380
PTP(DEGMan(OAc)4)3-2Py (30). Following procedure D, 28 was
reacted with BBr3 (0.05 mL, 0.48 mmol, 30 equiv) in dry CH2Cl2 (2.5
mL) to afford compound 29, which was directly engaged in procedure
G with Cs2CO3 (1.07 g, 3.28 mmol, 45 equiv) and [(2-bromoethoxy)-
O-ethoxy]-O-2′,3′,4′,6′-tetraacetyl-α-D-mannose (547 mg, 1.1 mmol,
15 equiv). The reaction was catalyzed with KI (3.6 mg, 0.022 mmol,
0.3 equiv). The expected compound was isolated by recrystallization
from CH2Cl2/n-heptane (35 mg, 20% yield) as a dark blue powder:
UV−vis in CH2Cl2 λmax nm (ε mM−1 cm−1) 450 (210.9), 581 (10.9),
1
638 (41.3); H NMR (CDCl3, 300 MHz) 9.71 (s, 2H), 8.92 (s, 2H),
8.78 (s, 4H), 8.58 (s, 4H), 8.10 (d, J = 7.7 Hz, 6H), 7.95 (d, J = 7.6
Hz, 2H), 7.53 (d, J = 7.4 Hz, 4H), 7.42−7.14 (m, 15H), 6.98 (d, J =
16.1 Hz, 2H), 5.49−5.28 (m, 9H), 4.99 (s, 3H), 4.41−3.89 (4m, 36H),
2.18 (s, 9H), 2.14 (s, 9H), 2.04 (s, 9H), 2.00 (s, 9H), −2.27 (s, 2H);
13C NMR (CDCl3, 75.3 MHz) 171.1, 170.7, 170.5, 170.4, 170.1, 170.0,
169.8, 158.7, 150.1, 147.2, 147.1, 144.8, 135.6, 134.3, 132.8, 132.4,
131.5, 128.2, 124.8, 124.5, 124.1, 120.7, 118.5, 113.0, 99.5, 97.8, 96.8,
92.2, 70.4, 70.0, 69.6, 69.5, 69.2, 68.5, 68.0, 67.7, 67.5, 67.0, 66.2, 63.6,
62.5, 30.9, 21.0, 20.8, 20.7; MS (MALDI-TOF) m/z [M]+ calcd for
C126H127N7O36 2314.84, found 2314.75.
PTPM(DEGMan(OAc)4)3-2Py (31). Compound 30 (35 mg, 15 μmol,
1 equiv) was metalated following procedure F in the presence of zinc
acetate (14 mg, 76 μmol, 5 equiv). Compound 31 was isolated
quantitatively as a blue powder and used without purification: UV−vis
1
(75.9), 446 (359.3), 584 (30.1), 634 (82.6); H NMR (CDCl3, 300
MHz) 9.62 (d, J = 4.6 Hz, 2H), 8.84 (d, J = 4.6 Hz, 2H), 8.72 (dd, J1 =
12.3 Hz, J2 = 4.6 Hz, 4H), 8.15 (d, J = 8.5 Hz, 2H), 8.08 (d, J = 8.4 Hz,
4H), 7.94 (d, J = 8.5 Hz, 2H), 7.70 (t, J = 7.3 Hz, 9H), 7.44 (d, J = 8.5
Hz, 4H), 7.35 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.5 Hz, 4H), 6.95−6.76
(m, 8H), 6.25 (d, J = 4.6 Hz, 2H), 5.80 (d, J = 16.0 Hz, 2H), 3.26 (s,
6H); 13C NMR (CDCl3, 75.3 MHz) 151.5, 150.8, 149.3, 146.8, 142.5,
135.1, 133.8, 125.8, 124.8, 123.8, 99.2, 52.7, 33.5, 29.6; MS (MALDI-
TOF) m/z [M]+ calcd for C78H53N9Zn 1179.37, found 1179.40;
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HRMS (MALDI-TOF) m/z [M]+ calcd for C78H53N9Zn 1179.3715,
found 1179.3752.
respectively and antibiotics in humidified atmosphere under 5% CO2
in air at 37 °C.
Dark Toxicity and Phototoxicity Assays. 5 × 105 Y79 cells and
5 × 104 HT-29 cells per well in 1 mL of appropriate culture medium
were seeded into 24-well plates. After 5 h of incubation at 37 °C for
Y79 and 24 h for HT-29, compounds 4 and 5, in DMSO solution at
10−3 or 10−4 M, were added in the dark at a final concentration
ranging from 0.75 to 7.5 μM in duplicate. Two plates of each cell line
were realized, one for dark toxicity and the other for phototoxicity.
Control cells received 7.5 μL of DMSO free of dye. After 24 h of
incubation with compound at 37 °C in the dark, the cells were washed
with phosphate buffered saline (PBS) and fresh medium free of drug
was added. Illumination was performed for 27 min and 20 s (5 J/cm2)
through the bottom of the 24-well plates using a “light box” made of
six Phillips TL 13W tubes (emission wavelength between 400 and 800
nm), leading to a final irradiance of 3 mW/cm2. Plates were left to
incubate at 37 °C in the dark for 3 days before evaluation of the cell
viability by determination of mitochondrial activity using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT,
Sigma) assay. At the time of counting, 50 μL of a MTT (5 mg/mL)
solution was added to each well. After 30 min of incubation and
removal of the medium, formazan crystals were taken up with 600 μL
of DMSO and absorbance at 562 nm was measured with a Fluostar
microplate reader. Survival rate was expressed as ratio of the
absorbance of treated and illuminated cells to 0.75% DMSO treated
controls. IC50 (half maximal inhibitory concentration) values
corresponding to the concentration of drug leading to 50% of survival,
were calculated from the dose−response curves and are expressed in
μM.
PTPM(OTEG)-2Py (4). Following procedure A, 200 mg of 20 (142
μmol, 1.0 equiv) reacted with NaH (27 mg, 1.14 mmol, 8 equiv) and
diethyl (4-pyridinyl)methyl phosphonate (130 mg, 0.57 mmol, 4
equiv) in dry THF (5 mL). Silica gel chromatography eluted with
CH2Cl2/EtOH (99/1 to 95/5, v/v) followed by recrystallization from
CH2Cl2/MeOH afforded the title compound with 99% yield (220 mg)
as a dark blue powder: UV−vis in DMSO λmax nm (ε mM−1 cm−1) 451
(228.8), 582 (12.9), 632 (40.3); 1H NMR (CDCl3, 300 MHz) 9.67 (d,
J = 4.2 Hz, 2H), 8.88 (d, J = 4.1 Hz, 2H), 8.78 (s, 4H), 8.04 (d, J = 7.7
Hz, 6H), 7.81 (d, J = 7.9 Hz, 2H), 7.25−7.15 (m, 16H), 6.98 (m, 4H),
6.75 (d, J = 16.1 Hz, 2H), 6.38 (m, 6H), 4.37 (m, 6H), 4.01 (m, 6H),
3.84 (m, 6H), 3.76 (m, 6H), 3.72 (m, 6H), 3.60 (m, 6H), 3.40 (s,
9H); 13C NMR (CDCl3, 75.3 MHz) 158.3, 151.9, 150.9, 150.2, 149.8,
147.2, 146.6, 144.6, 135.7, 135.5, 133.2, 132.9, 132.7, 132.6, 131.4,
130.7, 130.1, 128.1, 124.0, 123.5, 121.3, 119.9, 112.3, 98.5, 95.3, 94.1,
70.2−67.6, 59.1; MS (MALDI-TOF) m/z [M]+ calcd for
C93H89N7O12Zn 1559.59, found 1559.59; HRMS (MALDI-TOF)
(m/z) [M]+ calcd for C93H89N7O12Zn 1559.5861, found 1559.5862.
PTPM(DEGMan(OH)4)3-2Py (5). A freshly prepared solution of
sodium methanolate in dry methanol (4 mL, 0.1 M, 400 μmol, 24
equiv) was added to a solution of 31 (40 mg, 17 μmol, 1 equiv) in
anhydrous DCM (4 mL). The solution was stirred at room
temperature for 1 h. Then IWT ion-exchange resin was added, and
the mixture was stirred carefully. After 30 min, the resin was filtered off
and washed with a mixture of pyridine and water (1/1, v/v). Solvents
were evaporated under reduced pressure. The reaction gave the title
compound with a quantitative yield (30 mg) as a dark blue powder:
UV−vis in DMSO λmax nm (ε mM−1 cm−1) 450 (159.2), 581 (5.8),
Cellular Uptake. Internalization of compounds 4 and 5 was
measured by flow cytometry using a FACSCalibur four color
instrument with CellQuest software. This FACSCalibur was equipped
with a 488 nm argon laser with 530 nm ( 15 nm, FL1-H), 585 nm
( 21 nm, FL2-H) band-pass fluorescence filters and 670 nm (>670
nm, FL3-H) long-pass filter and a 635 nm red diode laser with a 661
nm band-pass filter ( 8 nm, FL4-H).
1
639 (29.8); H NMR (pyridine-d5, 300 MHz) 10.26 (d, J = 4.4 Hz,
2H), 9.32 (d, J = 4.4 Hz, 2H), 9.17 (s, 4H), 8.70 (d, J = 4.0 Hz, 4H),
8.26 (d, J = 7.9 Hz, 6H), 8.18 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz,
4H), 7.60 (d, J = 10.5 Hz, 2H), 7.50 (d, J = 5.5 Hz, 4H), 7.41 (d, J =
7.7 Hz, 8H), 7.34 (d, J = 8.4 Hz, 4H), 7.29−7.13 (m, 2H), 5.50 (s,
3H), 5.29 (br s, 9H), 4.66 (m, 12H), 4.41 (m, 12H), 4.20 (m, 3H),
4.14−4.06 (m, 3H), 4.02 (s, 6H), 3.90 (m, 9H); 13C NMR (pyridine-
d5, 75.3 MHz) 171.2, 171.0, 159.6, 153.3, 153.2, 151.2, 148.1, 147.9,
145.6, 140.0, 133.8, 133.4, 133.2, 132.8, 131.6, 129.5, 126.4, 126.0,
125.5, 125.2, 121.7, 119.8, 113.7, 102.5, 100.1, 97.2, 94.9, 76.0, 74.6,
73.6, 72.6, 71.4, 70.5, 69.7, 68.6, 67.5, 65.3, 63.7, 62.3, 56.6; MS
(MALDI-TOF) m/z [M]+ calcd for C102H101N7O24Zn 1871.62, found
1871.74, 1894.73 [M + Na]+; HRMS (MALDI-TOF) (m/z) [M]+
calcd for C102H101N7O24Zn 1871.6189, found 1871.6199.
For Y79 cells and HT-29 cells, 5 × 106 cells/well in 2 mL were
seeded into 6-well plates with the appropriate culture medium
(supplemented with 20% FCS for Y79 and 10% for HT-29). After 24 h
of incubation, the compounds were added to a final concentration of
7.5 μM. Control cells were incubated with 15 μL of DMSO (0.75%).
After 24 h of incubation in the dark at 37 °C, the cells were harvested.
Y79 cells were centrifuged, washed and resuspended in 500 μL of PBS.
HT-29 cells were trypsinized, centrifuged, washed and resuspended in
500 μL of PBS. Cells were analyzed by flow cytometry with the
appropriate settings for each cell line. A least, fifty thousand cells were
acquired initially and cells visualized on the basis of size (forward
scatter) and granularity (side scatter). The debris and aggregates were
excluded. Results are expressed as the percentage of compound-
positive cells and the measurement of cellular uptake level using mean
(MFI) of each detector. The ratio of MFI was calculated by dividing
the value of treated cells by the one of control for each compound and
each cell line. Finally, the means of three independent experiments
were calculated.
Photophysics. Fluorescence quantum yields (ΦF) were measured
according to Crosby’s comparative method using Coumarin 153 in
EtOH (ΦF = 0.38) as reference.52 One-photon singlet oxygen (1O2)
generation was detected by its phosphorescence at 1270 nm through a
PTI S/N 1565 monochromator, and the emission was monitored by a
liquid nitrogen-cooled Ge-detector model (EO-817L, North Coast
Scientific Co). Excitation occurred with a Xe-arc; the light was
1
separated in a SPEX 1680, 0.22 μm double monochromator. The O2
quantum yields (ΦΔ) were calculated by a comparative method using
tetraphenylporphyrin in DCM (ΦΔ = 0.60) or Rose Bengal in DMF
(ΦΔ = 0.47) as a standard in the Crosby method. Characterization of
the two-photon absorption properties of the dyes was obtained after
two-photon-induced fluorescence (TPIF) measurements, with a
femtosecond Ti:sapphire laser source delivering 100 fs pulses at 76
MHz repetition rate over the spectral range from 770 to 900 nm. The
TPIF intensities of the samples were measured relative to a solution of
Fluorescein, the ratio of the fluorescent signals enabling further
determination of the 2PA cross section (δ) assuming equal one- and
two-photon fluorescence quantum yields.53
Cell Lines and Cell Culture Conditions. The human
retinoblastoma cell line Y79 and the human colorectal adenocarcinoma
cell line HT-29 were obtained from the American Type Culture
Collection. Y79 cells (cultured in suspension) and adherent HT-29
cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)
Glutamax supplemented with 20% and 10% fetal calf serum (FCS),
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of all new compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: philippe.maillard@curie.p-psud.fr. Tel: (+33) (0)1 69
86 31 71. Fax: (+33) (0)1 69 07 53 27.
■
Present Address
$Institut des Sciences Chimiques de Rennes, UMR CNRS
6226, IUT de Lannion, rue Edouard Branly, BP 30219, F-22302
Lannion Cedex, France.
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The Journal of Organic Chemistry
Article
(20) Oar, M. A.; Serin, J. M.; Dichtel, W. R.; Frec
Ohulchanskyy, T. Y.; Prasad, P. N. Chem. Mater. 2005, 17, 2267−
2275.
́
het, J. M. J.;
Notes
The authors declare no competing financial interest.
(21) Wen, Y.-N.; Song, W.-S.; An, L.-M.; Liu, Y.-Q.; Wang, Y-H.;
Yang, Y.-Q. Appl. Phys. Lett. 2009, 95, 143702−143704.
(22) Nickel, E.; Spangler, C. W. Rebane, A. K. Patent US6953570,
2005.
ACKNOWLEDGMENTS
■
We acknowledge CNRS, the “Programme Incitatif et
Cooper
Curie, and the nonprofit French organization “Ret
(http://www.retino-stop.fr) for their financial support and
Vincent Guerineau for MALDI-TOF analysis from ICSN-
́
atif Ret
́
inoblastome et Transcriptome” of Institut
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́
inostop”
́
CNRS, Gif sur Yvette, France. G.G., S.A., and F.P. acknowledge
INCa and Fondation Pierre-Gilles De Gennes for postdoctoral
fellowships, and F.H. thanks Paris-Sud University and ENS
Cachan for Ph.D. funding. We acknowledge Dr. Patrice Baldeck
and Jean Bernard from UMR-CNRS 6688 and Universite
Joseph Fourier, Grenoble, France, for the 2PA measurements
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