Design and photophysical properties of new RGD targeted tetraphenylchlorins and porphyrins

Article abstract of DOI:10.1016/j.tet.2008.01.142

The synthesis, characterization, fluorescence, and singlet oxygen quantum yields of tetraphenylporphyrin and tetraphenylchlorin coupled to RGD type peptide are reported. C-terminus protected RGD derivatives were synthesized in liquid phase at the gram scale via an efficient convergent process. C-terminus unprotected RGD derivatives were synthesized on solid support. The UV-vis, fluorescence, and singlet emission spectra showed that the photophysical properties of the photosensitizers were retained in all compounds and some of them are very promising for potential PDT applications.

Full text of DOI:10.1016/j.tet.2008.01.142

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3494e3504
www.elsevier.com/locate/tet
Design and photophysical properties of new RGD targeted
tetraphenylchlorins and porphyrins
a
Michel Boisbrun , Regis Vanderesse , Philippe Engrand , Alexis Olie , Sebastien Hupont ,
b
a
c
c
´
Jean-Bernard Regnouf-de-Vains , Celine Frochot
´
´
a
c,
*
´
a SRSMC, Nancy-Universite´, CNRS, GEVSM, 5, rue Albert Lebrun, F-54001 Nancy, France
b DCPR, Nancy-Universite´, CNRS, 1 rue Grandville, BP 20451, F-54001 Nancy, France
c LCPM, Nancy-Universite´, CNRS, 1 rue Grandville, BP 20451, F-54001 Nancy, France
Received 20 December 2007; accepted 31 January 2008
Available online 6 February 2008
Abstract
The synthesis, characterization, fluorescence, and singlet oxygen quantum yields of tetraphenylporphyrin and tetraphenylchlorin coupled to
RGD type peptide are reported. C-terminus protected RGD derivatives were synthesized in liquid phase at the gram scale via an efficient con-
vergent process. C-terminus unprotected RGD derivatives were synthesized on solid support. The UVevis, fluorescence, and singlet emission
spectra showed that the photophysical properties of the photosensitizers were retained in all compounds and some of them are very promising for
potential PDT applications.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Porphyrin; Chlorin; Photodynamic therapy; RGD peptide; Cyclic RGD peptide; Fluorescence spectroscopy; Singlet oxygen
1. Introduction
For this purpose, we have devised a synthetic route to por-
phyrin derivatives designed for targeting tumors and more spe-
cifically neovascularization that feeds cancer cells. We have
initiated a program of research aimed at developing photosen-
sitizers targeting endothelial cells. One strategy consists in tar-
geting vascular endothelial growth factor (VEGF) receptor or
co-receptor6 since VEGF is one of the most potent direct-
acting angiogenic proteins known,7 up-regulated in the major-
ity of cancers. Another strategy aims at targeting avb3 integrin,
a heterodimeric transmembrane glycoprotein receptor, which
is overexpressed in actively proliferating endothelial cells, in
and around tumor tissues.8
In the mid 1980s, the H-Arg-Gly-Asp-OH (RGD) tripeptide
sequence has been identified as a major cell adhesion recogni-
tion motif.9 This finding, together with the subsequent discov-
ery of the integrin gene family of cell adhesion receptors that
recognize the RGD motif has established a highly conserved
cell adhesion system regulating cell migration, growth, differ-
entiation, and apoptosis.10 Not surprisingly, to date a large va-
riety of RGD derivatives11 and mimetics12 have been designed
Photodynamic therapy (PDT) is a promising treatment for
a variety of diseases mainly in anticancer field.1 PDT requires
a photosensitizer (PS), light, and molecular oxygen, whose
combined action results in the formation of reactive oxygen
species, singlet oxygen being the main mediator of cellular
death.2 One of the main drawbacks of the PS is their lack of
selectivity toward cancer cells. Until now, most of the efforts
in the development of tumor targeting photosensitizers have
focused on the targeting of markers overexpressed by tumor
cells themselves.3 Nevertheless, the vascular effect is thought
to play a major part in the eradication of some vascularized
tumors by PDT.4 Therefore, anti-neovascular PDT could be
proposed for a large number of vascularized tumors and is
a promising strategy in cancer treatment that has received con-
siderable attention in the recent years.5
* Corresponding author. Tel.: þ33 3 83 17 51 15; fax: þ33 3 83 37 81 20.
E-mail address: celine.frochot@ensic.inpl-nancy.fr (C. Frochot).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.142

M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
3495
that are studiedas therapeutics for thetreatment of thrombosis,13
tumor metastasis,14 and infectious diseases,15 or serve as adhe-
sive devices in tissue engineering, tissue renewal16 or tissue
attachment to prostheses,17 as well as in tumor diagnosis18 or
photodynamic therapy,19 or inhibition of secondary cataract.20
For the latter, we have shown that the protection of terminal
NH2e or/and eCOOH groups of RGD by simple derivations
such as acetyl, Boc, or benzylamide resulted in interesting evo-
lutions of the inhibitory effect, versus unprotected RGD, and
versus the RGDS tetrapeptide, which is commonly used as ref-
erence compound in the literature.9,21
reagents 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium-
tetrafluoroborate (TBTU) (3 equiv), 1-hydroxybenzotriazole
(HOBt) (3 equiv), and N,N-diisopropylethylamine (DIEA)
(9 equiv) in dimethylformamide (DMF). During the photosensi-
tizer coupling step, light exposure was minimized by sealing the
reaction vessel in aluminum foil to limit the occurrence of un-
wanted side reactions. A standard cleavage with trifluoroacetic
acid(TFA)andscavengersaffordedthecrudepeptidesorthepep-
tide-conjugatedphotosensitizer. Bothisomersofthechlorin,aris-
ingfromtheasymmetricalcharacterofthemolecule, couldnotbe
separated due to their similar retention time on reverse phase
HPLC (15.1 and 15.5 min).
We have developed rationalized and versatile procedures, to
produce these target RGD derivatives. Classical solid-phase
synthesis has been used to synthesize the RGD peptide and
to couple it to a porphyrin or a chlorin (compounds 5 and 6,
Scheme 1). A possible way of improving the affinity for
avb3 integrin is to protect the C-terminal part of the peptide.
In liquid phase, a C-benzylamide RGD peptide has been syn-
thesized and coupled to monocarboxylic tetraphenylporphyrin
succinimidyl ester (TPP-COOSu 1) and chlorin (Chl-COOSu
2) via two different spacers (Ahx 3 and PEG-Su 4), to evaluate
the influence of the type of the linker on the phototoxicity of
the new targeted photosensitizers for PDT applications (com-
pounds 19, 20a,b, 25 and 26, Scheme 3). Another way to im-
prove the affinity of the targeted photosensitizer for avb3
integrin is to use a cyclic peptide since it is generally accepted
that cyclic RGD peptides display a high activity compared to
the linear counterparts. Then, a cyclic peptide has been synthe-
sized on solid phase and coupled to a chlorin (compound 33).22
The tripeptide 15 was synthesized in liquid phase at the
gram scale using a convergent process resulting in an overall
yield of 51% (Scheme 2).
The condensation of Boc-Arg-OH 7 with HCl$H-Gly-OBzl
8 using DCC/HOBt afforded Boc-Arg-Gly-OBzl, which was
subsequently hydrogenated to give Boc-Arg-Gly-OH 9 with
an overall yield of 83% in a one-pot procedure. Coupling
Boc-Asp(OBzl)-OH 11 with benzylamine 10 with DCC/
HOBt afforded Boc-Asp(OBzl)-NHBzl 12 in 93% yield. Re-
moval of Boc protecting group with HCl gave HCl$H-Asp-
(OBzl)-NHBzl 1320a in 93% yield. Coupling of 9 with 13
was achieved by using the same reagents, combined with trib-
utylamine instead of the commonly used triethylamine. By this
way the resulting hydrochloride salt, soluble enough in or-
ganic solvents, was easily removed by triturating the crude
mixture, while the peptide adhered to the flask. The purifica-
tion process of 14 was achieved by column chromatography.
Lyophilization of the aqueous solution of 14 allowed the full
removal of residual organic solvents. Thus, 14 was obtained
with a yield of 68%. Classical treatment of 14 by trifluoroace-
tic acid afforded 15 in 91% yield.
2. Results and discussion
2.1. Syntheses
Before coupling to the photosensitizers 1 and 2, 15 had to be
linked with apolar (3) or polar (4) linkers (Fig. 1, Scheme 3).
For this purpose, 6-aminohexanoic acid 3 (Ahx) was
N-protected to give 16, and then activated to afford Z-Ahx-
OSu 17, as previously described.24 The latter was condensed
with 15 and subsequently hydrogenated to give Ahx-RGD-
NHBzl 18 in 40% overall yield. Coupling reaction with TPP-
COOSu 1 or Chl-COOSu 2 led to the desired products 19,
20a, and 20b in 35, 20, and 20% yield, respectively. It is note-
worthy that the two isomers 20a and 20b could be separated
by HPLC since their retention time on reverse phase HPLC
were different enough (15.9 and 16.9 min). In a similar way,
Aiming at comparing their photophysical properties, we
synthesized some RGD targeted photosensitizers for potential
PDT applications.
Compounds 5 and 6 were synthesized on a multichannel
peptide synthesizer, according to a classical Fmoc/tBu solid-
phase methodology (Scheme 1).
Assembly of the protected peptide chains was carried out us-
ing the in situ neutralization protocol described previously.23
Double coupling was performed using a threefold excess of
N-Fmoc-amino acid (except during the photosensitizer coupling
stage: threefold excess and single coupling) and activation
i, ii
i, ii
H
Asp(OtBu)
H
Gly
Arg(Pbf) Gly
Asp(OtBu)
H
Asp(OtBu)
i
PS Arg(Pbf) Gly Asp(OtBu)
iii
5 PS = porphyrin
6 PS = chlorin
PS Arg Gly Asp
H
Scheme 1. Reagents and conditions: (i) amino acid or PS, TBTU, HOBt, DIEA (ii) TFA; (iii) TFA, PhOH, EDT, thioanisole, H2O.

3496
M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
O
O
H
N
C
NH
O
Boc
HN
OH
ClH ,H2N
BocHN
+
O
BocHN
11
ii
H2N
+
7
8
12
O
10
O
OBzl
OBzl
H2N
NH,HCl
i
iii
O
O
H
N
OH
O
Boc
HN
N
H
C
NH
HCl,H2N
O
OBzl
H2N
NH, HCl
9
13
ii
O
O
O
O
H
H
N
H
N
N
H2N
Boc
N
H
NH
O
N
H
NH
O
iv
O
O
14
15
OBzl
OBzl
HN
H2N
HN
NH, HCl
NH, 2CF3COOH
H2N
Scheme 2. Reagents and conditions: (i) (1) DCC, HOBt; (2) H2, Pd/C; (ii) DCC, HOBt; (iii) HCl; (iv) TFA.
2,20-(ethylenedioxy)bis-ethylamine 21 was mono-N-protected
to give 22, which was further reacted with succinic anhydride
to give Z-PEG-Su 23, as previously described.25 The latter
was condensed with 15 with DCC/HOBt and tributylamine,
and subsequently hydrogenated to give PEG-Su-RGD-NHBzl
24 in 28% overall yield. The latter was too polar to be
O
O
O
i
ii
N
O
Bzl
Bzl
H2N
CO2H
C
O
O
N
H
CO2H
O
N
H
O
3
16
17
O
O
H
N
H
N
iii
iv
H2N
N
H
NH
O
PS-Ahx-RG D-NHBzl
1 or 2
O
O
19 PS = porphyrin
OH
20a PS = chlorin (isomer a)
20b PS = chlorin (isomer b)
HN
18 H2N
NH,CF3COOH
O
O
H
N
vi
v
O
NH2
Bzl
O
CO2H
Bzl
O
O
NH2
H2N
O
O
N
H
O
N
H
O
O
21
22
23
O
O
H
H
N
O
N
O
CONH
vii
H2N
N
NH
O
iv
1or 2
H
PS-PEG-Su-RGD-NHBzl
O
O
24
OH
25 PS = porphyrin
26 PS = chlorin
HN
H2N
NH,CF3COOH
Scheme 3. Reagents and conditions: (i) ZCl, NaOH; (ii) DCC, HOSu; (iii) (1) 15, TEA; (2) H2, Pd/C; (iv) PS-OSu 1 or 2, DIEA; (v) ZOSu, NaOH; (vi) succinic
anhydride; (vii) (1) 15, DCC, HOBt, Bu3N; (2) H2, Pd/C.

M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
3497
NH
N
NH
N
N
O
O
O
O
C
C
H
O
N
O
N
N
H
HN
HN
H
O
O
H
1
2
O
NH
CO2H
H2N
CO2H
H2N
O
4
O
3
Figure 1.
analytically pure after normal phase column chromatography.
Crude 24 was then directly coupled with TPP-COOSu 1 or
Chl-COOSu 2 to afford the corresponding PS-PEG-Su-RGD-
NHBzl 25 and 26 in 58 and 60% yield, respectively.
For the synthesis of cyclo[RGDfK(CO-Chl)] 33, we fol-
lowed and adapted the improved synthesis based on Kessler’s
procedure developed by Dai et al.26 We chose to couple first
in liquid phase the monocarboxylic tetraphenylchlorin succin-
imidyl ester (Chl-COOSu) 2 to the amine function of the
lysine lateral side chain (Scheme 4) as it has been already
described.19c
This site of attachment on the peptide was chosen because the
lateral side chain ofthelysineisnotessential for activityandthus,
the coupling preserves the specificity of the cyclopeptide foravb3
integrin and can also act as a spacer. Coupling of Fmoc-Lys-OH
with N-hydroxysuccinimide activated Chl-COOSu 2 afforded
Fmoc-Lys(CO-Chl)-OH28, which canbecoupledonsolid-phase
support to the resin-Gly-Arg(Pbf)-NH2 (Scheme 5).
N
NH
N
O
NH
N
NH
O
O
i
ii
C
COOH
C
H
H
NH
N
H
N
H
H
H
H
O
HN
N
H
N
H
HN
HN
(CH2)4
O
H
H
H
COOH
Fmoc-NH
28
27
2
Scheme 4. Reagents and conditions: (i) HOSu; (ii) Fmoc-Lys-OH$HCl, TEA.
ii
i
H-Asp(OtBu)-phe-Lys-Arg(Pbf)-Gly
AcOH,H-Asp(OtBu)-phe-Lys-Arg(Pbf)-Gly OH
H-Gly
CO-Chl
31
29
CO-Chl
30
iii
O
Arg Gly
NH phe
iv
O
Asp
N
HN
Asp(OtBu)-phe-Lys-Arg(Pbf)-Gly
C
N
H
NH
N
CO-Chl
32
(Lys)
33
Scheme 5. Reagents and conditions: (i) amino acid, TBTU, HOBt, DIEA; (ii) CH3COOH, TFE, CH2Cl2; (iii) T3P, AcOEt; (iv) TFA, EDT, phenol, thioanisole, H2O.

3498
M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
The linear RGDfK 31 peptide was cleaved from the resin
spacer. The structure of the linker influenced the absorption
properties, and the compound with the PEG arm displayed an
higher extinction coefficient value at 650 nm (2373 Mꢀ1 cmꢀ1
vs 1266 Mꢀ1 cmꢀ1).3c To our knowledge, no systematic study
has been done concerning the influence of the spacer on the
photochemical and biological properties of the photosensi-
tizers. In a recent publication, Maillard et al.27 reported the syn-
thesis and in vitro photoactivity of a series of glycol conjugated
porphyrins and chlorins on the 179 retinoblastoma cell line. The
sugar component was directly attached to the p-hydroxyphenyl
moiety or through a diethylene glycol linker for one porphyrin
TPP(p-O-Deg-O-b-D-GluOH)3. No difference in absorption
was observed. Nevertheless, the porphyrin monosaccharide
conjugates, in which the sugar and porphyrin moieties were
separated by diethylene glycol spacer, were essentially non
cytotoxic and displayed good to excellent phototoxicity.
The fluorescence emission spectra of all the compounds
display two bands at around 650 and 717 nm. As previously
observed,3b fluorescence quantum yields are higher for chlor-
ins than for porphyrins, 20e29% against 5e9%, respectively.
This is not in favor of good photosensitizers since we expect
the compounds to produce singlet oxygen.
Singlet oxygen quantum yields F(O2) were determined us-
ing tetraphenylporphyrin (TPP) as reference solution (F(1O2)
[TPP]¼0.68, in toluene) and were estimated from 1O2 lumines-
cence at 1272 nm. All the compounds present good to excellent
singlet oxygen quantum yields compatible for a use in PDT. If
compound 26 is the best candidate for PDTapplication in terms
of absorption at 650 nm, it is not the best for singlet oxygen
production. Porphyrins reveal better singlet oxygen production
than their chlorin homologues as previously observed.3b
Surprisingly, both isomers 20a and 20b exhibit different
photophysical properties that is to say a difference of 10%
for singlet oxygen quantum yield and 20% in absorption. To
the best of our knowledge, it is the first time that both isomers
have been separated by HPLC and it has been shown that the
photophysical properties are slightly different.
without affecting other protecting groups with a mixture of
acetic acid, 2,2,2 trifluoroethane (TFE), and DCM (1:1:3).
The head-to-tail cyclization was performed by treatment
with 1-propane-phosphonic acid cyclic anhydride (T3P) to af-
ford the chlorin coupled to the protected cyclic peptide 32.
The other protecting groups of the above cyclic peptide were
removed with trifluoroacetic acid (TFA) leading to the desired
product 33 with an overall yield of 5%.
Compound 33 was obtained with a final purity greater than
95%, as assessed by analytical RP-HPLC. Two isomers, corre-
sponding to the reduction of a double bond on either opposing
side of the tetrapyrrolic macrocycle could be observed by
RP-HPLC but not separated (17.7 and 18.2 min). Identities
of the compounds were confirmed by MALDI-TOF mass spec-
trometry and NMR experiments.
2.2. Photophysical properties
Table 1 shows the compared photophysical properties of the
RGD targeted photosensitizers synthesized by our group.
Room temperature absorption and fluorescence emission
spectra of targeted porphyrins 19 and 25 and chlorins contain-
ing 20a,b and 26 were recorded in ethanol. All the compounds
show the Soret band (414<lmax<420 nm) and the four Q bands
around 515, 545, 596, and 650 nm common for porphyrins and
chlorins in the UVevis region. To have a better penetration of
the light in the tissues, the QI band (around 650 nm) is used for
PDT applications. It is interesting to notice that, at this specific
wavelength, the porphyrinated compounds 5, 19, and 25 present
the same low absorption whatever the spacer is. In contrast, we
can notice a high influence of the type of the arm on the extinc-
tion coefficient of the chlorin containing ones. Compound 26
presents the best absorption, which is around four times better
than the chlorin with the Ahx spacer 20 and almost five times
better than the chlorin without any spacer 6. This may be ex-
plained by the increased solubility of the compound due to
the PEG spacer and this is in good agreement with previous
study. 4-Carboxyphenylporphyrin was coupled to folic acid
via 1,6-diaminohexane or 2,20-(ethylenedioxy)-bis(ethylamine)
3. Conclusion
New targeted photosensitizers for anti-vascular photody-
namic therapy have been synthesized with success in good
yields. The photophysical properties of the photo-activable moi-
ety are conserved after the coupling of the vector. In conclusion,
if chlorins present the best absorption at 650 nm, porphyrins
have very good singlet oxygen quantum yields. A PEG spacer
increases the hydrophily of the targeted photosensitizers. This
preliminary invitro results concerning chlorin or porphyrin cou-
pled to RGD are very promising.19c The invitro study of the new
compounds is under progress and will be described elsewhere.
Table 1
Photophysical properties of photosensitizers-RGD compounds
Compound
3Soret
lSoret
lQI
Ffe
F1O2
a
b
c
3QI
d
f
5
136,000
19,600
54,352
39,220
32,817
85,876
52,232
53,300
415
415
414
418
420
414
415
415
1520
1800
1668
7809
5938
1673
8323
3400
649
649
650
650
651
647
651
649
0.05
0.18
0.09
0.25
0.23
0.09
0.29
0.18
0.60
0.50
0.77
0.72
0.60
0.58
0.57
0.82
6
19
20a
20b
25
26
33
a
4. Experimental
Molar extinction coefficient of Soret band (103 cm3 molꢀ1).
Soret band wavelength.
b
c
d
e
f
4.1. General
Molar extinction coefficient of QI band.
QI wavelength.
Fluorescence quantum yield.
Unless otherwise stated, reagents were purchased from
chemical companies and used without prior purification.
Singlet oxygen formation quantum yield.

M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
3499
Reagent grade solvents were used as received. 5-(4-Carboxy-
4.2. Syntheses
phenyl)-10,15,20-triphenylchlorin (TPC) was purchased from
Frontier Scientific (Logan, Utah). 5-(4-Carboxyphenyl)-
10,15,20-triphenylporphyrin was synthesized as previously
described.3b The Fmoc-Asp(OtBu)-Wang and H-Gly-2-chloro-
trityl resins, and 9-fluorenyl-methoxy-carbonyl (Fmoc)-ami-
noacids were from Senn Chemicals International (Dielsdorf,
Switzerland). Thin layer chromatography (TLC) was carried
out on Merck silica gel 60 F254 plates (Merck Chimie
S.A.S., Fontenay Sous Bois, France) and developed with the
appropriate solvents. The TLC spots were visualized either
by UV light or by heating plates sprayed with a solution of
phosphomolybdic acid (5% ethanolic solution). Chromatogra-
phy column was carried out on Merck silica gel (40e63 mm;
230e400 mesh ASTM). Assembly of compounds 5, 6, and
33 was carried out on a multichannel peptide synthesizer
PSP 4000, according to a classical Fmoc/tBu solid-phase
methodology using the in situ neutralization protocol.23 For
the attachment of each amino acid, double couplings (20
and 40 min, respectively) were performed using a threefold
excess of N-Fmoc-amino acid and activation reagents 2-
(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumtetrafluoro-
borate (TBTU) (3 equiv), 1-hydroxybenzotriazole (HOBt)
(3 equiv), and N,N-diisopropylethylamine (DIEA) (9 equiv)
in dimethylformamide (DMF). Monitoring of the reaction
was performed by the 2,4,6-trinitrobenzenesulfonic acid test.
For the attachment of the photosensitizer, only one coupling
step using twofold excess was applied. During the photosensi-
tizer coupling stage and all the next steps, light exposure was
minimized by sealing the reaction vessel in aluminum foil to
limit the occurrence of unwanted side reactions. Before cleav-
age, the peptide-resin was washed extensively with dichloro-
methane and dried in vacuo. A semi-preparative Alltech
Apollo C18, 5 mm column (250ꢁ10 mm ID, Alltech, Lokeren,
Belgique) was used for purification. Fluorescence detection
was optimized with excitation and emission wavelengths of
415 and 650 nm, respectively, and coupled with UVabsorption
(350 nm). All the targeted photosensitizers were eluted with
a gradient of methanoleH2O (75:25, v/v) for 15 min, followed
by 100% methanol for the last 15 min. Room temperature and
a flow-rate of 4.0 mL minꢀ1 were maintained throughout the
purification. A volume of 1 mL of the samples in methanol
was injected into the column. 1H and 13C NMR spectra were re-
corded on a Bruker Advancer 300 (300.130 and 75.475 MHz,
respectively), or on a Bruker DRX 400 (400.130 and
100.612 MHz, respectively). Multiplicities are reported as fol-
low: s¼singlet, t¼triplet, q¼quadruplet, and m¼multiplet.
Mass spectra analyses (MALDI-TOF) were carried out on
a Bruker Reflex IV time-of-flight mass spectrometer (Bruker-
Daltonic, Bremen, Germany) equipped with the SCOUT 384
probe ion source, and electospray (ESI-MS) on a Platform
Micromass apparatus. Melting points (ꢂC, uncorrected) were
determined on a Electrothermal 9100 Capillary apparatus. In-
frared spectra were recorded on a Bruker Vector 22 apparatus
(KBr, n in cmꢀ1), and elemental analyses were performed on
a Thermofinnigan FlashEA 1112 apparatus, at the Service
Commun de Microanalyse, Nancy.
4.2.1. 5-(4-Carboxy-RGD-phenyl)-10,15,20-triphenyl-
porphyrin 5 and 5-(4-carboxy-RGD-phenyl)-10,15,20-
triphenylchlorin 6
The synthesis was performed using the preloaded Fmoc-
Asp(OtBu)-Wang (capacity, 0.79 mmol/g) on a 0.15 g scale.
The side chains of arginine and aspartic acid were, respec-
tively, protected by Pbf (2,2,5,7,8-pentamethylchroma-6-sulfo-
t
nyl) group and Bu (tert-butyl). The successive coupling of
Fmoc-Gly-OH (106 mg) and Fmoc-Arg(Pbf)-OH (230 mg)
in the presence of TBTU (114 mg), HOBt (54 mg), and
0.18 mL of DIEA in 5 mL of DMF was achieved. After the re-
moval of the Fmoc group from arginine, the photosensitizer
was coupled by using the same protocol. The photosensiti-
zerepeptideeresin was washed with CH2Cl2 (6ꢁ5 mL) and
then dried in vacuo overnight. A standard cleavage with a mix-
ture of 0.75 g of crystalline phenol, 0.25 mL of 1,2-ethane-
dithiol, 0.5 mL of thioanisole, 0.5 mL of deionized H2O, and
10 mL of trifluoroacetic acid (TFA) for 1.5 h afforded the tar-
geted photosensitizer, which was lyophilized. The compounds
were purified by RP-HPLC. After removal of the solvents, the
purified compounds were lyophilized and analyzed by 1H
NMR and mass spectroscopy.
4.2.2. 5-(4-Carboxy-RGD-phenyl)-10,15,20-triphenyl-
porphyrin 5
Yield 55%. 1H NMR (300 MHz, DMSO-d6): d ꢀ2.91 (s, 2H,
NH-pyrrole), 1.58 (m, 3H, 2g,1bArg), 1.70 (m, 1H, bArg),
2.54, 2.74 (ABX, JAB¼16.2 Hz, JAx¼5.7 Hz, JBx¼7.9 Hz,
2H, bAsp), 3.12 (m, 2H, dArg), 3.59, 3.98 (ABX, JAB¼16.9 Hz,
JAx¼5.8 Hz, JBx¼5.3 Hz, 2H, aGly), 4.28 (br t, 1H, aArg), 7.22
(m, 1H, 3NHArg), 8.05 (br d, 1H, NHArg), 8.25 (br t, 1H,
NHGly), 7.23e8.83 (m, 27H, ArH, pyrrole). MS (MALDI-
TOFMS): m/z found: 987.78 [MþH]þ.
4.2.3. 5-(4-Carboxy-RGD-phenyl)-10,15,20-triphenyl-
chlorin 6
Yield 58%. 1H NMR (300 MHz, DMSO-d6): d ꢀ1.52,
ꢀ1.58 (s, 2H, NH-pyrrole), 1.50 (m, 3H, 2g,1bArg), 1.72
(m, 1H, bArg), 2.58, 2.82 (ABX, JAB¼16.1 Hz, JAx¼5.6 Hz,
JBx¼8.0 Hz, 2H, bAsp), 3.11 (m, 2H, dArg), 3.62, 4.02
(ABX, JAB¼16.9 Hz, JAx¼5.7 Hz, JBx¼5.4 Hz, 2H, aGly),
4.10 (br t, 1H, aAsp), 4.14 (m, 4H, CH2-chlorin), 4.32 (br t,
1H, aArg), 7.23 (br t, 1H, 3NHArg), 7.98 (br d, 1H, NHArg),
8.20 (br t, 1H, NHGly), 7.23e8.57 (m, 23H, ArH, pyrrole).
MS (MALDI-TOFMS): m/z found: 989.34 [MþH]þ.
4.2.4. tert-Butyloxycarbonylarginylglycine
hydrochloride 9
A solution of tert-butyloxycarbonylarginine 7 (1.163 g,
4.24 mmol), dicyclohexylcarbodiimide (874 mg, 4.24 mmol),
hydroxybenzotriazole hydrate (649 mg, 4.24 mmol), and gly-
cine benzylester hydrochloride 8 (854 mg, 4.24 mmol) in
DMF (30.00 mL) was stirred at room temperature under argon
for 12 h. The precipitate was filtered and washed with EtOAc
(3ꢁ5 mL). The solution was concentrated under reduced

3500
M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
pressure to give a yellow gum. Deprotection of the benzyles-
ter: the residue was dissolved in MeOH (50 mL) and to the re-
sulting solution was added 10% Pd/C (250 mg). The mixture
was stirred under H2 at atmospheric pressure at room temper-
ature for 12 h. The suspension was filtered through CeliteÒ
and the solvent was evaporated. The resulting oil was dis-
solved in 20 mL of MeOH and the title compound was precip-
itated as a gum by the addition of Et2O (300 mL). The
suspension was let for half an hour, then the mixture of sol-
vents was removed, while the compound sticked to the flask.
The dissolution/precipitation process was renewed three
more times, using 25 mL of MeOH for the last dissolution.
The final residue was carefully dried under reduced pressure
to give 1.332 g (3.54 mmol, 83.5% yield) of white material.
Mp 108e110 ꢂC. IR (KBr): n¼3343, 2979, 1165, 1525,
[MþNa]þ, 451.21 [MþK]þ. Anal. Calcd for C23H28N2O5
(Boc-Asp(OBzl)-NHBzl: 412.48): C, 66.97; H, 6.84; N, 6.82.
Found: C, 67.13; H, 7.06; N, 6.93.
4.2.6. Benzylaspartamide-b-benzylester hydrochloride 13
A stirred solution of 12 (4.739 g, 11.49 mmol) in EtOAc
(150 mL) was cooled in an ice bath. Hydrogen chloride gas
was let bubble in the solution for 2 h, then stirring was main-
tained for 5 h at the same temperature. The resulting suspen-
sion was filtered and washed with Et2O (2ꢁ15 mL) to give
3.383 g of white powder. Filtration and washing of the filtrate
with Et2O (2ꢁ5 mL) gave another amount (0.329 g) of the
same material. Total yield of 13: 3.712 g (93% yield). Mp
157 ꢂC. IR (KBr): n¼1730, 1666. 1H NMR (400 MHz,
DMSO-d6): d 3.01 (m, 2H, bAsp), 4.18 (t, J¼6.9 Hz, 1H,
aAsp), 4.33 (d, J¼5.9 Hz, 2H, benzylamide CH2), 5.15 (s,
2H, benzylester CH2), 7.24e7.40 (m, 10H, ArH), 8.43 (br s,
3H, NH3), 9.06 (t, J¼5.8 Hz, 1H, benzylamide NH). 13C
NMR (100 MHz, DMSO-d6): d 36.0 (CH2 of Asp), 43.3 (ben-
zylamide CH2), 49.8 (CH of Asp), 67.1 (benzylester CH2),
127.8 (Ph CH), 128.2 (Ph CH), 128.9 (Ph CH), 129.0 (Ph
CH), 129.1 (Ph CH), 129.3 (Ph CH), 136.5 (Ph Cquat), 139.3
(Ph Cquat), 168.0 (ester CO), 169.9 (amide CO). ESI-MS
(pos. mode): m/z 313.23 [MþH]þ. Anal. Calcd for
C18H21N2O3Cl (H-Asp(OBzl)-NHBzl$HCl: 348.82): C,
61.98; H, 6.07; N, 8.03. Found: C, 61.76; H, 6.17; N, 8.00.20b
1
1368, 1251, 1165. H NMR (400 MHz, DMSO-d6): d 1.38
t
(s, 9H, Bu), 1.50 (m, 3H, 2g,1bArg), 1.67 (m, 1H, bArg),
3.08 (m, 2H, dArg), 3.70, 3.80 (ABX, JAB¼17.6 Hz,
JAx¼5.7 Hz, JBx¼5.7 Hz, 2H, aGly), 3.96 (m, 1H, aArg),
6.93 (d, J¼8.2 Hz, 1H, NHBoc), 7.00e7.75 (br s, 4H, guani-
dinium), 7.78 (m, 1H, NHArg), 8.16 (t, J¼7.5 Hz, 1H,
NHGly), 12.60 (COOH). 13C NMR (100 MHz, DMSO-d6):
d 25.3 (gArg), 28.4 (CH3 Boc), 29.3 (bArg), 40.5, 40.9
(dArg, aGly), 53.8 (aArg), 78.3 (Cquat Boc), 155.5 (CO
Boc), 157.2 (guanidinium), 171.4, 172.4 (CO Arg, COOH
Gly). ESI-MS (pos. mode): m/z 354.2 [MþNa]þ, 332.1
[MþH]þ. Anal. Calcd for C13.25H27N5.25O5Cl (Boc-RG-
OH$HCl, 0.25MeOH: 375.85): C, 42.34; H, 7.24; N, 18.64.
Found: C, 42.22; H, 7.31; N, 18.73.
4.2.7. tert-Butyloxycarbonylarginylglycylbenzylaspart-
amide-b-benzylester hydrochloride 14
A solution of tert-butyloxycarbonylarginylglycine hydro-
chloride (1.385 g, 3.68 mmol), dicyclohexylcarbodiimide
(760 mg, 3.68 mmol), hydroxybenzotriazole hydrate (564 mg,
3.68 mmol), and benzylaspartamide-b-benzylester hydrochlo-
ride 1320b (1.285 g, 3.68 mmol) in DMF (50.00 mL) was stirred
at room temperature under argon for 0.5 h. To the resulting sus-
pension was added tributylamine (877 mL, 3.68 mmol) and the
stirring was maintained for 12 h. The precipitate was filtered
and washed with EtOAc (3ꢁ5 mL). The solution was concen-
trated under reduced pressure to give a yellow gum, which
was dissolved in CH2Cl2 (70 mL). The desired compound
was precipitated as a gum by addition of Et2O (200 mL). The
suspension was let for half an hour, then the mixture of solvents
was removed, while the compound sticked to the flask. The dis-
solution/precipitation process was renewed once and column
chromatography (eluent: CH2Cl2/MeOH, 19:1 then 18:2, then
17:3) afforded a white solid material, which was dissolved in
CH3CN (30 mL) and H2O (30 mL), and then lyophilized to
give 1.672 g (68% yield) of white powder. Mp 100e110 ꢂC.
IR (KBr): n¼3324, 2978, 1735, 1662, 1527, 1366, 1251,
4.2.5. tert-Butyloxycarbonylbenzylaspartamide-b-
benzylester 12
A solution of Boc-Asp(OBzl)-OH (4.000 g, 12.37 mmol),
DCC (2.552 g, 12.37 mmol), and HOBt hydrate (1.894 g,
12.37 mmol) in THF (100 mL) was stirred at room tempera-
ture for 15 min. Benzylamine (1.49 mL, 13.60 mmol) was
then added to the resulting suspension and stirring was main-
tained for 5 h. The mixture was filtered and washed with
EtOAc (2ꢁ10 mL). The solution was concentrated under re-
duced pressure and to the residue was added CH2Cl2
(50 mL). The insoluble material was filtered off and washed
with CH2Cl2 (2ꢁ5 mL). The filtrate was concentrated under
reduced pressure and the residue was purified by column chro-
matography (eluent: CH2Cl2, then CH2Cl2/MeOH, 99:1) to
give 4.739 g of white solid (93% yield). Mp 91 ꢂC. IR
1
(KBr): n¼1687, 1667. H NMR (400 MHz, CDCl3): d 1.44
t
(s, 9H, Bu), 2.78 (dd, J1¼17.4 Hz, J2¼5.7 Hz, 1H, bAsp),
3.13 (dd, J1¼17.2 Hz, J2¼4.4 Hz, 1H, bAsp), 4.38e4.55 (quar-
tet dþm, 3H, aAspþbenzylamide CH2), 5.12 (s, 2H, benzyles-
ter CH2), 5.69 (br d, 1H, NHBoc), 6.81 (br d, 1H, benzylamide
NH), 7.26e7.40 (m, 10H, ArH). 13C NMR (100 MHz, CDCl3):
d 28.7 (tBu CH3), 36.5 (CH2 of Asp), 43.9 (benzylamide CH2),
51.2 (CH of Asp), 67.3 (benzylester CH2), 81.0 (tBu Cquat),
127.9 (Ph CH), 127.9 (Ph CH), 128.7 (Ph CH), 128.8 (Ph
CH), 129.0 (Ph CH), 129.1 (Ph CH), 135.8 (Ph Cquat), 138.3
(Ph Cquat), 156.0 (carbamate CO), 171.0 (ester CO), 172.2 (am-
ide CO). ESI-MS (pos. mode): m/z 413.24 [MþH]þ, 435.24
1
1168, 738, 699. H NMR (400 MHz, DMSO-d6): d 1.38 (s,
9H, tBu), 1.50 (m, 3H, 2g,1bArg), 1.67 (m, 1H, 1bArg), 2.68,
2.88 (ABX, JAB¼16.2 Hz, JAx¼5.8 Hz, JBx¼7.8 Hz, 2H,
bAsp), 3.08 (m, 2H, dArg), 3.72, 3.79 (ABX, JAB¼16.7 Hz,
JAx¼5.2 Hz, JBx¼5.4 Hz, 2H, aGly), 3.95 (m, 1H, aArg),
4.28 (d, J¼5.9 Hz, 2H, NHCH2Ph), 4.72 (m, 1H, aAsp), 5.09
(s, 2H, OCH2Ph), 7.01 (d, J¼7.4 Hz, 1H, NHArg), 7.02e7.65
(m, 14H, Haromþguanidinium), 8.19 (br t, 1H, NHGly), 8.33

M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
3501
(d, J¼8.2 Hz, 1H, NH-Asp), 8.49 (br t, 1H, NHCH2Ph). 13C
NMR (100 MHz, DMSO-d6): d 25.2 (gArg), 28.4 (CH3 Boc),
29.0 (bArg), 36.5 (bAsp), 40.5 (dArg), 42.3 (NHCH2Phþ
aGly), 49.7 (aAsp), 54.0 (aArg), 65.8 (OCH2Ph), 78.4 (Cquat
Boc), 126.8, 127.2, 128.0, 128.1, 128.3, 128.5 (CH Ph),
136.2, 139.3 (Cquat Ph), 155.6 (CO Boc), 157.1 (guanidinium),
169.0, 170.2 (2 pics), 172.5 (CO Gly, bCO Asp, CO Arg, CO
Asp). ESI-MS (pos. mode): m/z 626.31 [MþH]þ. Anal. Calcd
for C31H44N7O7Cl (662.18): C, 56.22; H, 6.70; N, 14.81.
Found: C, 55.95; H, 6.77; N, 14.63.
for 12 h. The suspension was filtered through CeliteÒ, and the
solvent was evaporated to give a white solid, which was dis-
solved in H2O (10 mL) and lyophilized to afford 154 mg
(40% yield over the two steps) of white powder. Mp 222 ꢂC.
IR (KBr): n¼3378, 3070, 2941, 1662 (br peak), 1538, 1393,
1200, 1130, 722. 1H NMR (400 MHz, DMSO-d6): d 1.28
(m, 2H, CH2 Ahx), 1.47 (m, 7H, 2CH2 Ahxþ2g,1bArg),
1.99 (m, 1H, 1bArg), 2.12 (t, J¼7.2 Hz, 2H, CH2CO Ahx),
2.23, 2.64 (ABX, JAB¼16.1 Hz, JAx¼3.4 Hz, JBx¼5.1 Hz,
2H, bAsp), 2.72 (m, 2H, CH2NH Ahx), 2.94 (m, 1H, dArg),
3.11 (m, 1H, dArg), 3.49, 3.83 (ABX, JAB¼16.9 Hz,
JAx¼5.8 Hz, JBx¼5.3 Hz, 2H, aGly), 4.13, 4.39 (ABX,
JAB¼15.6 Hz, JAx¼6.8 Hz, JBx¼5.5 Hz, 2H, CH2Ph), 4.28
(m, 2H, aAspþaArg), 7.08 (br s, 2H, guanidinium), 7.25 (s,
5H, Ph), 7.57 (br t, 1H, NHCH2Ph), 7.77 (br s, 3H, guanidi-
nium), 7.94 (d, J¼7.6 Hz, 1H, NHArg), 8.86 (br t, 1H,
NHGly), 8.98 (d, J¼8.0 Hz, 1H, NH-Asp), 10.28 (br s, 1H,
COOH). 13C NMR (100 MHz, DMSO-d6): d 24.8, 25.0, 25.8
(CH2 Ahx, gArg), 27.1 (bArg), 29.8 (CH2 Ahx), 35.0
(CH2CO Ahx), 37.8 (bAsp), 39.1 (CH2NH2), 40.6 (d Arg),
42.3 (aGly), 43.4 (NHCH2Ph), 50.2 (aAsp), 51.9 (aArg),
117.6 (q, J¼297 Hz, CF3), 126.8, 126.9, 128.3 (CH Ph),
139.6 (Cquat Ph), 157.8 (guanidinium C), 158.5 (q, J¼31 Hz,
CF3COOH), 168.7, 171.6, 172.5, 174.1, 176.0 (CO Gly,
bCO Asp, CO Arg, CO Ahx, CO Asp). ESI-MS (pos.
mode): m/z 571.1 [MþNa]þ, 549.1 [MþH]þ, 275.3
[MþH]2þ. Anal. Calcd for C27H45N8O10F3 (H-Ahx-RGD-
NHBzl, CF3COOH, 2H2O): C, 46.41; H, 6.49; N, 16.04.
Found: C, 46.15; H, 6.25; N, 15.95.
4.2.8. Arginylglycylbenzylaspartamide-b-benzylester
bis-trifluoroacetate monohydrate 15
To a solution of 14 (1.622 g, 2.45 mmol) in CH2Cl2
(160 mL) was added trifluoroacetic acid (53 mL). The solution
was stirred 0.75 h at room temperature, then concentrated in
vacuo. To the residue was added Et2O (210 mL). The resulting
white precipitate was filtered, washed with Et2O (3ꢁ30 mL),
and dried to give a white powder, which was dissolved in
H2O (45 mL). The solution was lyophilized to give 1.714 g
(91% yield) of white powder. Mp 65e70 ꢂC. IR (KBr):
1
n¼3423, 3068, 2929, 1735, 1672, 1204, 1135, 723. H NMR
(400 MHz, DMSO-d6): d 1.54 (m, 2H, 2gArg), 1.71 (m, 2H,
2bArg), 2.68, 2.86 (ABX, JAB¼16.1 Hz, JAx¼5.7 Hz,
JBx¼8.2 Hz, 2H, bAsp), 3.11 (m, 2H, dArg), 3.70e3.92 (m,
3H, aArg, aGly), 4.28 (d, J¼5.7 Hz, 2H, NHCH2Ph), 4.73
(m, 1H, aAsp), 5.1 (s, 2H, OCH2Ph), 6.80e7.60 (m, 14H,
Haromþguanidinium), 7.70 (br t, 1H, 3NHArg), 8.15 (br s,
3H, NH3 Arg), 8.47 (d, J¼8.0 Hz, 1H, NH-Asp), 8.54 (t,
J¼5.8 Hz, 1H, NHCH2Ph), 8.71 (br t, 1H, NHGly). 13C
NMR (100 MHz, DMSO-d6): d 24.9 (gArg), 29.2 (bArg),
37.3 (bAsp), 41.0 (dArg), 42.7 (aGly), 43.1 (NHCH2Ph),
50.4 (aAsp), 52.6 (aArg), 66.6 (OCH2Ph), 118.0 (q,
J¼297 Hz, CF3), 127.6, 127.9, 128.7, 128.9, 129.1, 129.3
(CH Ph), 136.9, 140.0 (Cquat Ph), 157.8 (guanidinium C),
159.6 (q, J¼31 Hz, CF3COOH), 169.1, 169.6, 170.8, 170.9
(CO Gly, bCO Asp, CO Arg, CO Asp). ESI-MS (pos.
mode): m/z 526.3 [MþH]þ, 436.3 [MꢀBnþH]þ. Anal. Calcd
for C30H39N7O10F6 (HRGD(OBn)NHBn, 2CF3COOH, H2O:
771.68): C, 46.69; H, 5.09; N, 12.71. Found: C, 46.80; H,
5.20; N, 12.60.
4.2.10. N-Succinimidylarginylglycylbenzylaspartamide-
2,20-(ethylenedioxy)bis(ethylamine) trifluoroacetate 24
Coupling: to a solution of 23 (148 mg, 0.39 mmol) in DMF
(10 mL) were added 15 (299 mg, 0.39 mmol), DCC (80 mg,
0.39 mmol), and HOBt hydrate (59 mg, 0.39 mmol). The solu-
tion was stirred for 0.3 h at room temperature, then tributyl-
amine (0.092 mL, 0.39 mmol) was added, and stirring was
maintained for 12 h. The resulting suspension was filtered
and the precipitate was washed with CH2Cl2 (2ꢁ4 mL). The
mixture of solvent was evaporated and the residue was dis-
solved in CH2Cl2 (20 mL). The desired compound was precip-
itated as a gum by addition of Et2O (20 mL). The suspension
was let for 1 h, then the mixture of solvents was removed,
while the compound sticked to the flask. The dissolution/
precipitation process was renewed once and column chroma-
tography (eluent: CH2Cl2/MeOH, 19:1 then 18:2, then 17:3)
afforded 110 mg of white solid material. Deprotection: to a so-
lution of the latter compound in MeOH (12 mL) was added
10% Pd/C (40 mg). The mixture was stirred under H2 at atmo-
spheric pressure at room temperature for 12 h. The suspension
was filtered through CeliteÒ, and the solvent was evaporated to
give a white solid, which was dissolved in H2O (10 mL) and
lyophilized to afford 83 mg (28% yield over the two steps)
of white powder. This crude compound was used without fur-
ther purification. Mp 98 ꢂC. ESI-MS (pos. mode): m/z 705.0
[MþK]þ, 688.0 [MþNa]þ, 666.30 [MþH]þ, 352.7
4.2.9. 6-Aminohexanoylarginylglycylbenzylaspartamide
trifluoroacetate dihydrate 18
To a solution of 15 (426 mg, 0.55 mmol) in DMF (20 mL)
was added a solution of 6-(benzyloxycarbonylamino)hexanoic
acid succinimidyl ester (200 mg, 0.552 mmol) in CH2Cl2
(20 mL) and triethylamine (0.077 mL, 0.552 mmol). The mix-
ture was stirred at room temperature for 12 h and concentrated
under reduced pressure. To the residue was added H2O
(10 mL). The resulting gel was sonicated, filtered, and washed
with water (3ꢁ10 mL). After drying (Na2SO4), it was purified
by column chromatography (eluent: CH2Cl2/MeOH, 95:5 then
90:10, then 85:15) to give 222 mg of white solid material. De-
protection: to a solution of the latter compound in MeOH
(20 mL) was added 10% Pd/C (55 mg). The mixture was
stirred under H2 at atmospheric pressure at room temperature
[MþK]2þ, 333.8 [MþH]2þ
.

3502
M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
4.2.11. 5-(4-Carboxyphenyl succinimidyl ester)-10,15,20-
triphenylporphyrin 1 and 5-(4-carboxyphenyl succinimidyl
ester)-10,15,20-triphenylchlorin 2
guanidinium), 7.86 (br d 1H, NHArg), 8.83 (br t, 1H, NHGly),
8.98 (br d, 1H, NH-Asp). MS (MALDI-TOFMS): m/z found:
1191.196 [MþH]þ.
1
In the dark under a nitrogen atmosphere, N-hydroxysuccin-
imide (0.23 mmol) and dicyclohexylcarbodiimide (DCC)
(0.23 mmol) were added to a solution of 5-(4-carboxyphenyl)-
10,15,20-triphenylporphyrin or 5-(4-carboxyphenyl)-10,15,20-
triphenylchlorin (0.23 mmol) in CH2Cl2 (6 mL). The mixture
was stirred 4 h at room temperature. The solvent was evaporated
and the crude material was purified by column chromatography
using EtOH/CH2Cl2 4:96 (v/v) as the eluent. The fractions were
tested by TLC and the pure compound was isolated as a purple
solid (112 mg, 65% for 1, 127 mg, 73% for 2).
Compound 1: 1H NMR (300 MHz, DMSO-d6): d ꢀ2.75 (s,
2H, NH-pyrrole), 2.97 (s, 4H, CH2 Su), 7.27e8.91 (m, 27H,
ArH, pyrrole).
Compound 2: 1H NMR (300 MHz, DMSO): d ꢀ1.50,
ꢀ1.65 (s, 2H, NH-pyrrole), 2.99 (s, 4H, CH2 Su), 4.17 (s,
4H, CH2-chlorin), 7.26e8.85 (m, 27H, ArH, pyrrole).
Compound 20b: H NMR (DMSO-d6, 300 MHz): except
the NH-pyrrole (d ꢀ1.54), the spectrum is superimposable to
that of 20a. MS (MALDI-TOFMS): m/z 1191.325 [MþH]þ.
4.2.14. 5-(4-Carboxy-PEG-Su-RGD-NHBzl-phenyl)-
10,15,20-triphenylporphyrin 25
The coupling was performed as describe for 19 (vide supra)
on a 0.1 mmol scale with an overall yield of 58%.
1H NMR (300 MHz, DMSO-d6): d ꢀ2.91 (s, 2H, NH-pyr-
role), 1.45 (m, 3H, 2g,1bArg), 1.97 (m, 1H, 1bArg), 2.18e
2.64 (m, 6H, Su, bAsp), 2.88 (br t, 2H PEG), 3.12 (m, 2H,
dArg), 3.31 (td, J¼5.0 Hz, 2H PEG), 3.43, 3.78 (m, 8H,
PEG, 2H aGly), 4.12, 4.43 (ABX, JAB¼15.7 Hz, JAx¼6.8 Hz,
JBx¼5.4 Hz, 2H, CH2Ph), 4.28 (m, 2H, aAspþaArg), 7.13 (s,
1H, 3NHArg), 7.19 (s, 5H, Ph), 7.23e8.83 (m, 27H, ArH, pyr-
role), 7.12 (br t, 1H, NHCH2Ph), 7.77 (br s, 3H, guanidinium),
7.28 (d, J¼7.6 Hz, 1H, NHArg), 8.75 (br t, 1H, NHGly), 8.95
(d, J¼8.0 Hz, 1H, NH-Asp). MS (MALDI-TOFMS): m/z
1307.274 [MþH]þ.
4.2.12. 5-(4-Carboxy-Ahx-RGD-NHBzl-phenyl)-10,15,20-
triphenylporphyrin 19
General procedure for the coupling of the photosensitizers:
compound 18 (51 mg, 0.093 mmol) was added to a solution of
5,10,15,tri(p-tolyl)-20-(p-carboxyphenyl succinimidyl ester)-
porphyrin 1 (70 mg, 0. 0.093 mmol) in DMF/CH2Cl2 (v/v
10 mL) and DIEA (16 mL, 0.093 mmol). The mixture was
stirred at room temperature for five days and concentrated un-
der reduced pressure. The crude product was lyophilized and
purified by RP-HPLC to give 38.70 mg (35% yield). 1H
NMR (300 MHz, DMSO-d6): d ꢀ2.90 (s, 2H, NH-pyrrole),
1.09 (m, 2H, CH2 Ahx), 1.38 (m, 7H, 2CH2 Ahxþ2g,1bArg),
1.79 (m, 1H, 1bArg), 2.28 (br s, 2H, CH2CO Ahx), 2.36, 2.68
(ABX, JAB¼16.1 Hz, JAx¼3.4 Hz, JBx¼5.1 Hz, 2H, bAsp),
3.12 (m, 2H, dArg), 3.41, 3.79 (ABX, JAB¼16.3 Hz,
JAx¼7.8 Hz, JBx¼5.3 Hz, 2H, aGly), 3.55 (m, 2H, CH2NH
Ahx), 4.19 (m, 1H, aArg), 4.23 (d, J¼5.6 Hz, 2H, CH2Ph),
4.29 (m, 1H, aAsp), 7.13 (br s, 1H, 3NHArg), 7.24e8.84
(m, 27H, ArH, pyrrole), 7.57 (br t, 1H, NHCH2Ph), 7.77 (br,
3H, guanidinium), 7.89 (d, J¼7.5 Hz, 1H, NHArg), 8.79 (br
t, 1H, NHGly), 8.83 (br, 1H, NH-Asp). MS (MALDI-
TOFMS): m/z 1190.421 [MþH]þ.
4.2.15. 5-(4-Carboxy-PEG-Su-RGD-NHBzl-phenyl)-
10,15,20-triphenylchlorin 26
The coupling was performed as describe for 19 (vide supra)
on a 0.1 mmol scale with an overall yield of 60%.
1H NMR (300 MHz, DMSO-d6): d ꢀ1.58. ꢀ1.53 (s, 2H,
NH-pyrrole), 1.33 (m, 3H, 2g,1bArg), 1.87 (m, 1H, 1bArg),
2.25e2.69 (m, 6H, Su, bAsp), 3.36 (m, 2H, dArg), 3.62,
3.89 (ABX, JAB¼16.7 Hz, JAx¼5.8 Hz, JBx¼5.3 Hz, 2H,
aGly), 4.13 (s, 4H, CH2-chlorin), 4.15, 4.48 (ABX,
JAB¼15.6 Hz, JAx¼6.9 Hz, JBx¼5.5 Hz, 2H, CH2Ph), 4.41
(m, 2H, aAspþaArg), 7.18 (br s, 7H, NHCH2Ph, 3NHArg,
Ph), 7.20e8.84 (m, 23H, ArH, pyrrole), 7.28 (d, J¼7.6 Hz,
1H, NHArg), 8.75 (br t, 1H, NHGly), 7.77 (br s, 3H, guanidi-
nium), 8.98 (d, J¼8.0 Hz, 1H, NH-Asp). MS (MALDI-
TOFMS): m/z calculated 1307.48; found: 1308.221
[MþH]þ. MS (MALDI-TOFMS) m/z 1308.221 [MþH]þ.
4.2.16. 2-(9H-Fluoren-9-ylmethoxy-carbonylamino)-6-
[5,10,15,tri(p-tolyl)-20-(p-carboxyamino)-chlorin]-
hexanoic acid (Fmoc-Lys(CO-Chl)-OH) 28
4.2.13. 5-(4-Carboxy-Ahx-RGD-NHBzl-phenyl)-10,15,20-
triphenylchlorin 20a and 20b
In the dark under a nitrogen atmosphere, to a solution of
81 mg of Fmoc-Lys-OH$HCl (0.20 mmol) in a minimum of
DMF were added N-hydroxysuccinimide activated chlorin 2
(152 mg, 0.20 mmol) and triethylamine (0.20 mmol, 28.3 mL)
in 10 mL CH2Cl2. After been stirred at ambient temperature
for 24 h, the solvent was evaporated and the crude material
was purified by column chromatography using EtOH/CH2Cl2
5:95 (v/v) as the eluent. The fractions were tested by TLC
and the pure compound was isolated as a purple solid by
column chromatography (152 mg, 75%).
1H NMR (300 MHz, DMSO-d6): d ꢀ1.52, ꢀ1.58 (s, 2H,
NH-pyrrole), 1.57 (m, 2H, gLys), 1.75 (m, 2H, dLys), 1.85,
2.00 (m, 2H, bLys), 3.57 (m, 1H, 3Lys), 4.11 (s, 4H,
CH2-chlorin), 4.14 (m, 1H, CH-Fmoc), 4.40 (m, 1H, aLys),
The coupling was performed as describe for 19 (vide supra)
on a 0.1 mmol scale with an overall yield of 40% for the mix-
ture of 20a and 20b.
1
Compound 20a: H NMR (DMSO-d6, 300 MHz): d ꢀ1.52
(s, 2H, NH-pyrrole), 1.26 (m, 2H, CH2 Ahx), 1.45 (m, 7H,
2CH2 Ahx, 2g,1bArg), 1.98 (m, 1H, 1bArg), 2.13 (br s, 2H,
CH2CO Ahx), 2.39, 2.70 (ABX, JAB¼16.1 Hz, JAx¼3.4 Hz,
JBx¼5.1 Hz, 2H, bAsp), 3.09 (m, 2H, dArg), 3.43, 3.73
(ABX, JAB¼16.2 Hz, JAx¼7.7 Hz, JBx¼5.4 Hz, 2H, aGly),
3.57 (m, 2H, CH2NH Ahx), 4.25 (m, 4H, aArg, aAsp,
CH2Ph), 7.13 (br s, 1H, 3NHArg), 7.24e8.84 (m, 27H, ArH,
pyrrole), 7.59 (br t, 1H, NHCH2Ph), 7.80 (br s, 3H,

M. Boisbrun et al. / Tetrahedron 64 (2008) 3494e3504
3503
4.36 (s, 2H, CH2-Fmoc), 5.76 (m, 1H, NH-Lys), 6.70 (m, 1H,
zNH-Lys), 7.18e8.82 (m, 31H, ArH, pyrrole, Fmoc).
4.4. Determination of singlet oxygen quantum yield (F(1O2))
Excitation occurred with a Xe-arc, the light was separated in
a SPEX 1680, 0.22 mm double monochromator. The detection at
1270 nm was done 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.). The
absorbance of the reference solution (Bengal pink in EtOH)
Ff(1O2)¼0.6829 and the sample solution (at 515 nm) was set
equal (between 0.2 and 0.5) by dilution.
4.2.17. Cyclo[RGDfK(CO-Chl)] 33
The synthesis was performed using the preloaded H-Gly-2-
chlorotrityl PS resin (capacity: 0.85 mmol/g) on a 0.115 g
scale. The side chains of arginine and aspartic acid were, re-
t
spectively, protected by Pbf and Bu groups. The successive
coupling of Fmoc-Arg(Pbf)-OH (190 mg), Fmoc-Lys-
(CO-Chl)-OH 28 (152 mg), Fmoc-(D)Phe-OH (114 mg), and
Fmoc-Asp(OtBu)-OH (121 mg) in the presence of TBTU
(94 mg), HOBt (45 mg), and 0.15 mL of DIEA were achieved.
After the final removal of the Fmoc group, the peptide-resin
was washed with CH2Cl2 (6.5 mL) and then dried in vacuo
overnight. The linear H-RGDfK(CO-Chl)-OH peptide 31
was cleaved from the resin without affecting other protecting
groups with 10 mL of a mixture of acetic acid, 2,2,2 trifluoro-
ethane (TFE) and CH2Cl2 (1:1:3) for 1 h at room temperature.
The resin was washed three times with acetic acid and then
lyophilized. The head-to-tail cyclization was performed by
slowly adding a solution of the linear peptide acetate salt 31
in 20 mL of CH2Cl2 to a solution of 50% 1-propane-phos-
phonic acid cyclic anhydride (T3P) in EtOAc (3 mL), triethyl-
amine (3 mL), and DMAP (10 mg) in 400 mL of CH2Cl2.
After stirring overnight, the reaction mixture was concentrated
and purified by chromatography (MeOH/AcOEt, 10:90) to af-
ford 115 mg of the chlorin coupled to the protected cyclic pep-
tide 32. A standard cleavage with a mixture of 0.75 g of
crystalline phenol, 0.25 mL of 1,2-ethanedithiol, 0.5 mL of
thioanisole, 0.5 mL of deionized H2O, and 10 mL of trifluoro-
acetic acid (TFA) for 1.5 h afforded the crude peptide 33,
which was lyophilized and was purified by RP-HPLC to
give 56 mg (45% yield). 1H NMR (300 MHz, DMSO-d6):
d ꢀ1.53, ꢀ1.58 (s, 2H, NH-pyrrole), 1.20 (m, 2H, dLys),
1.42 (m, 2H, gArg), 1.53, 1.72 (m, 2H, bArg), 1.55 (m, 2H,
dLys), 1.80 (m, 2H, bLys), 2.40, 2.81 (m, 2H, b(D)Phe),
2.84, 2.94 (m, 2H, bAsp), 3.12 (m, 2H, dArg), 4.01e4.15
(m, 4H, aLys, aGly, aArg), 4.52 (br d, 1H, aAsp), 4.70 (br
d, 1H, a(D)Phe), 4.13 (m, 4H, CH2-chlorin), 6.77 (m, 1H,
zNH-Lys), 7.48 (br t, 1H, 3NHArg), 7.65 (br d, 1H, NHArg),
8.05 (br d, 1H, NH-Asp), 8.08 (br d, 1H, (D)Phe-NH), 8.12
(br d, 1H, NH-Lys), 8.43 (br t, 1H, NHGly), 7.19e8.84 (m,
30H, ArH, pyrrole). MS (MALDI-TOFMS): m/z 1246.33
[MþH]þ. (MALDI-TOFMS): m/z 1246.33 [MþH]þ.
Acknowledgements
The authors thankfully acknowledge Dr. Marc Dodeller for
the MALDI-TOF mass spectrometry analysis and M-.J. Belgy
for her kind help in HPLC purifications.
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