Folic acid conjugates with photosensitizers for cancer targeting in photodynamic therapy: Synthesis and photophysical properties

Article abstract of DOI:10.1016/j.bmc.2016.10.004

Recent researches in photodynamic therapy have focused on novel techniques to enhance tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic acid could allow more effective targeting of folate receptors which are

Full text of DOI:10.1016/j.bmc.2016.10.004

Accepted Manuscript
Folic acid conjugates with photosensitizers for cancer targeting in photodynamic
therapy: synthesis and photophysical properties
Aurélie Stallivieri, Ludovic Colombeau, Gulim Jetpisbayeva, Albert
Moussaron, Bauyrzhan Myrzakhmetov, Philippe Arnoux, Samir Acherar, Régis
Vanderesse, Céline Frochot
PII:
S0968-0896(16)30911-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.10.004
BMC 13331
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 July 2016
14 September 2016
6 October 2016
Please cite this article as: Stallivieri, A., Colombeau, L., Jetpisbayeva, G., Moussaron, A., Myrzakhmetov, B.,
Arnoux, P., Acherar, S., Vanderesse, R., Frochot, C., Folic acid conjugates with photosensitizers for cancer targeting
in photodynamic therapy: synthesis and photophysical properties, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.10.004
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Graphical Abstract
Leave this area blank for abstract info.
Folic acid conjugates with photosensitizer for
cancer targeting in photodynamic therapy:
synthesis and photophysical properties
Aurélie Stallivieri, Ludovic Colombeau, Gulim Jetpisbayeva, Albert Moussaron, Bauyrzhan Myrzakhmetov,
Philippe Arnoux, Samir Acherar, Régis Vanderesseand Céline Frochot
a LRGP, UMR-CNRS 7274, and b LCPM, UMR-CNRS 7375, Lorraine-Université, 1 rue Grandville, BP 20451, 54001 Nancy Cedex, France
O
COOH
no linker or
″
Photosensitizer
(Porphyrin or Chlorin)
!
O
N
N
H
H
N
O
N
N
O
N
H
O
HN
H2N
N
H
Folic acid
OEG

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Folic acid conjugates with photosensitizers for cancer targeting in photodynamic
therapy: synthesis and photophysical properties
Aurélie Stallivieria, Ludovic Colombeaua, Gulim Jetpisbayevaa, Albert Moussarona, Bauyrzhan
Myrzakhmetova, Philippe Arnouxa, Samir Acherarb, Régis Vanderesseb and Céline Frochota,*
a Laboratoire Réactions et Génie des Procédés, UMR-CNRS 7274, Lorraine-Université, 1 rue Grandville, BP 20451, 54001 Nancy Cedex, France
b Laboratoire de Chimie Physique macromoléculaire, UMR-CNRS 7375, Lorraine-Université, 1 rue Grandville, BP 20451, 54001 Nancy Cedex, France
ARTICLE INFO
ABSTRACT
Article history:
Received
Recent researches in photodynamic therapy have focused on novel techniques to enhance
tumour targeting of anticancer drugs and photosensitizers. Coupling a photosensitizer with folic
acid could allow more effective targeting of folate receptors which are over-expressed on the
surface of many tumour cells. In this study, different folic acid-OEG-conjugated
photosensitizers were synthesized, characterized and their photophysical properties were
evaluated. The introduction of an OEG does not significantly improve the hydrophilicity of the
FA-porphyrin. All the FA-targeted photosensitizers present good to very good photophysical
properties. The best one appears to be Ce6. Molar extinction coefficient, fluorescence and
singlet oxygen quantum yields were determined and were compared to the corresponding
photosensitizer alone.
Received in revised form
Accepted
Available online
Keywords:
Cancer targeting
Folic acid
Photodynamic therapy
Singlet oxygen
Photosensitizers
2016 Elsevier Ltd. All rights reserved
.
1. Introduction
Photodynamic therapy (PDT) combines a photoactivable molecule - the photosensitizer (PS) - with oxygen and light. When
photosensitizers are exposed to light of a specific wavelength, the PS is promoted to an excited singlet state then to an excited
triplet state that can transfer energy to oxygen, to form singlet oxygen. Singlet oxygen is highly reactive, toxic and can react with
various cellular constituents causing cell damage and death.
Specific targeting of therapeutic agents to human tissues, regardless of the application fields, remains a challenge for
biologists and chemists. These agents must avoid biological changes leading to rapid elimination from the body and, at the
cellular level, the active molecules must distinguish different types of cells, be internalized by passing through membranes and
reach their target, while phenomena resistances encountered in tumor cells, must be bypassed. Many researches’ have been
undertaken to improve the selectivity between different types of cells, to increase the bioavailability or give better properties of
penetration into cells. For cancer treatment, these strategies can be summarized in two approaches, passive targeting and active
targeting, which are sometimes combined to increase efficiency. Passive targeting focuses on the physiological differences
between normal and tumor environments. Tumor tissues possess vasculature with a permeability and increased retention, leading
to greater accumulation of nutrients necessary for angiogenesis. This property is called 'enhanced permeability and retention
"(EPR). [1]Passive targeting of tumor cells by PDT is to vectorize PSs through different formulation systems facilitating
transport and incorporation into cancer cells. Several types of vectors can be used: liposomes, nanoparticles and micellar
systems. [1]
A strategy for improving the efficiency of PDT is to increase the selectivity towards tumour cells in order to reduce the
adverse side-effects caused by normal cell injury. Active targeting is based on the interaction of a vector with a cell surface
marker (receptor or antigen) which is over-expressed onto tumour cells. Among the tumour targeting agents, numerous
biomolecules have been studied. These include peptides,[2-4] lipoproteins,[5, 6] saccharides[7] or antibodies.[8] Folic acid (FA)

is also a targeting agent which has been widely studied in the field of imaging or diagnostics[9-11] and for the treatment of
certain cancers.[2, 3, 12, 13]1
Numerous cancer cell lines over-express FA receptors because of their fast growth and cell division.[14] Indeed, the folate
receptors (FRs) are over-expressed on prostate, brain, lung, nose, ovary, colon, cancer cells[9, 10] and have very low expression
on normal cells.[15] This selective over-expression makes FRs interesting tumour targets when FA is conjugated to anticancer
molecules.[16]
By attaching a PS to FA, in 2005 our team demonstrated the selectivity of the targeted PS for the first time.[17] Since this
study, other groups have shown the potential effectiveness of FA for tumour-specific drug delivery in the PDT field. Among the
different carriers of folate are nanoparticles,[2, 18-22] liposomes,[6, 23, 24] micelles,[25, 26] quantum dots[27-29] or carbon
nanotubes and other nanocomposites.[22, 30-33] Some studies have focused on the PS conjugation to FA via a spacer. The linker
is often a polyethylene glycol (PEG) arm of variable length.[34-37] Stefflova et al. were the first to design folate targeted
photosensitizers using a peptide sequence as stable linker between FA and PS.[38] More recently, directly linked FA-PS
molecules were synthesized.[39, 40] In a recent review we collected the data concerning the use of FA for PDT applications
from the literature.[41]
We chose two different classes of PS: “synthetic” PSs i.e. a porphyrin and a chlorin, and “natural” PSs i.e protoporphyrin IX
(PpIX) and chlorin e6 (Ce6). The objectives of our research were the synthesis and photophysical characterization of PSs to
evaluate the influence of the nature of the PS and the addition of a spacer onto the photophysical properties of the conjugates
(Figure 1).
N
HN
N
NH
N
N
NH
N
NH
N
NH
N
N
N
N
O
O
NH2
NH
HN
OH
OH
HN
HN
HN
COOH
COOH
O
HOOC
OH
OH
O
TPP-COOH
PpIX
Ce6
TPC-COOH
TPP-NH2
1
3
5
2
4
no linker or
O
COOH
γ
Photosensitizer
(Porphyrin or Chlorin)
O
N α
H
H
N
O
O
N
N
O
N
H
HN
N
H
Folic acid
OEG
H2N
N
Figure 1. Folic acid conjugate
2. Chemistry
1.1. Synthesis
Our strategy for the synthesis of PS-FA conjugates 12-13 was divided into three main parts and is shown in Scheme 1: first,
the preparation of tetraphenylporphyrin monocarboxylic acid TPP-COOH (1) and tetraphenylchlorin monocarboxylic acid TPC-
COOH 2, second the addition of a small oligo(ethylene glycol), 2,2’-(ethylenedioxy)-diethylamine abbreviated OEG, third
coupling to FA to give the corresponding conjugates.
Tetraphenylchlorin monocarboxylic acid TPC-COOH 2 was synthesized using the Lindsey method[42] to produce first
porphyrin TPP-COOH 1 and Whitlock di-imide reduction of this last porphyrin[43] (Scheme 2). Activation of the carboxylic
function of the starting porphyrins was performed by esterification of these porphyrins with N-hydroxysuccinimide (NHS) in
dichloromethane with N,N'-dicyclohexylcarbodiimide (DCC) as catalyst. Good yields of TPP-NHS 6 and TPC-NHS 7 were
obtained - 75 % and 89 %, respectively.
———
1 * Corresponding author: address Email: celine.frochot@univ-lorraine.fr

X
O
N
O
O
PS
O
O
(i)
(ii)
PS
O
O
PS
OH
HN
O
PS = TPP-COOH
PS = TPC-COOH
8
9
X = NHBoc
PS = TPP-COOH
PS = TPC-COOH
1
2
PS = TPP-COOH
PS = TPC-COOH
6
7
(iii)
PS = TPP-COOH 10
PS = TPC-COOH 11
X = NH2
NH
2
N
(iv)
N
N
PS = TPPCOOH 12
PS = TPC-COOH
N
OH
13
HN
NH
O
O
HN
O
O
O
PS
O
HN
HO
Scheme 1. Synthesis strategy for PS-OEG-FA 12, 13. (i) NHS, DCC, CH2Cl2, 45 °C, overnight. (ii) OEG (N-Boc-
2,2´-(ethylenedioxy)diethylamine), THF, r.t., 18-24 h. (iii) TFA, r.t., 2h. (iv) FA, DCC, pyridine, DMSO, r.t., 24h
H
N
NH
N
N
O
4 eq.
3 eq.
1 eq.
OH
HN
O
O
(ii)
NH
N
N
O
(i)
(iii)
OH
HN
(ii)
NH
N
O
TPP-COOH
1
OH
HN
N
HO
O
TPC-COOH
2
Scheme 2. Synthesis of photosensitizers TPP-COOH 1 and TPC-COOH 2. (i) BF3.(OEt)2, CH2Cl2, reflux, overnight and then, p-
chloranil. (ii) p-TSH, K2CO3, pyridine, reflux, overnight. (iii) EtOAc, o-chloranil.
TPP- and TPC-OEG-NHBoc 8 and 9 were synthesized from TPP- and TPC-NHS 6 and 7 with N-Boc-2,2´-
(ethylenedioxy)diethylamine in THF at room temperature as previously described[17]. Yields were comprised between 70% and
80 %. For the purification of compounds 8 and 9 on silica gel columns, CH2Cl2/EtOH (or MeOH) was preferred to
acetone/CH2Cl2/hexane (at a ratio of 1:4:5) as we previously described[17] for similar compounds. A better separation of side-
products could be observed without using hexane. The Boc group was removed by using trifluoroacetic acid (TFA) to give the
amine photosensitizers 10 and 11. Finally, these amine porphyrins were coupled to DCC-activated FA in pyridine/DMSO to give
the conjugates 12 and 13 after purification by RP-HPLC.

To evaluate the influence of the nature and distance of the PS from FA on the photophysical properties, we synthesized a
porphyrin directly coupled to FA (Scheme 3). The 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (TPP-NH2 5) was coupled to
FA activated by DCC in pyridine/DMSO to give the conjugate 14 at 4 % yield after purification by RP-HPLC. This rather poor
yield can be attributed to the very low reactivity of the amine of the compound 5 and also to an inadequate choice of the coupling
reagent. Activation of this amine by isothiocyanate (NCS) may help to ameliorate the coupling of the TPP-NH2 5 with folic acid.
NH2
N
N
N
N
OH
HN
NH
N
NH
N
N
N
(i)
NH2
NH
O
HN
HN
HN
O
O
HO
TPP-FA 14
TPP-NH2 5
Scheme 3. Synthesis of TTP-FA 14: (i) FA, DCC, pyridine, DMSO, r.t., 48h.
Secondly, we shall report on our synthesis of “natural” photosensitizer-FA conjugates 19 and 20. As “natural” porphyrin
moieties, we used either PpIX 3 or Ce6 4, which are the most well-known natural PSs possessing carboxylic functions used in
vivo for clinical applications.
HN
N
N
N
N
N
HN
N
HN
N
HN
N
(i)
(ii)
(iii)
NH
NH
NH
NH
O
O
O
O
OH
OH
OH
OH
OH
O
O
NH
O
NH
O
NH
3
15
17
19
O
O
O
O
O
O
HN
NH2
O
O
NH
Boc
HN
COOH
HN
N
N
N
N
H2N
OH
HN
N
N
N
N
HN
N
HN
N
HN
N
(i)
(ii)
(iii)
N
NH
NH
NH
NH
OH
O
O
OH
O
OH
O
NH
OH
O
O
O
O
O
O
O
OH
NH
OH
NH
OH
OH
OH
O
4
16
18
20
O
O
O
O
O
O
HN
O
O
HN
H2N
Boc
HOOC
NH
NH
N
N
N
HO
N
NH2

Scheme 4. Syntheses of PpIX- OEG-FA 19 and Ce6- OEG-FA 20: (i) Spacer (N-Boc-2,2´-(ethylenedioxy)diethylamine), DCC,
HOBt, DMF, r.t., 24h. (ii) TFA, r.t., 2h. (iii) FA, DCC, pyridine, DMSO, r.t., 24h.
Protoporphyrin IX 3 or chlorin e6 4 (Scheme 4) reacted with N-Boc-2,2´-(ethylenedioxy)diethylamine in DMF in by adding
dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt). After purification through a silica gel column (eluent:
CH2Cl2/EtOH gradient 100:0 to 0:100 +1% Et3N), mono-protected OEG protoporphyrin IX 15 and mono-protected OEG chlorin
e6 conjugates 16 were obtained at 43% and 39% yields, respectively. The Boc group was removed by TFA and the expected
conjugates 19 and 20 were obtained at 53% and 45% yields respectively, by coupling with FA which was activated by DCC in
pyridine/DMSO after precipitation in cold diethyl ether followed by centrifugation.
All the yields are reported in Table 1.
Considering only the “synthesis” parameter, even if the coupling of FA to TPP-NH2 was merely one step, the best yield was
obtained for the two natural commercial PSs Ce6 and PpIX.
Table 1. Yield of all the steps, overall yield and number of steps for each compound
Commercial
or synthesized
PS-NHS PS-OEG-Boc
PS-OEG-NH2
PS- OEG-FA
Overall
Nb of steps
11
7
-
75
89
-
73
78
43
39
-
85
24
49
52
-
28
62
53
45
4
1.4
1
5
6
3
3
1
TPP-COOH
TPC-COOH
PpIX
11.2
9.1
4
Ce6
-
-
TPP-NH2
-
-
1.2. Hydrophobicity of molecules
To compare the hydrophobicity of PSs alone and that of the corresponding PSs after coupling with FA (PS-OEG-FA), we
looked at the retention time of each molecule in reverse phase HPLC on a C18 column using an acetonitrile/water gradient (10/90
% to 100/0 % in 25 min, 100/0% for 15 min, acetonitrile/water, v/v). The comparison of retention times and consequently
hydrophobicities are shown in figure 2. A long retention time indicates a high hydrophobicity and vice versa. As expected, the
more carboxylic functions presented by the compounds, the less hydrophobic they are. Indeed, the order of hydrophobicity is
Ce6 (3 carboxylic functions) < PpIX (2 carboxylic functions) < TPP-NH2 (1 amine function that might be protonated due to the
presence of 0.1 % TFA in the water) < TPP-COOH (1 carboxylic function) < TPC-COOH. When coupled to FA, all the
compounds have lower retention times than the same compounds without FA. The coupling of FA to TPP-NH2 or TPP-OEG
leads to compounds with the same retention time. It seems that the introduction of the short OEG unit does not significantly
decrease the hydrophobicity of the compound. This is probably due to the small size of the OEG. In a previous study [3], we
evaluated the influence of three spacers (aminohexanoic acid (Ahx), 1-amino-3,6-dioxaoctanoic acid (PEG9), and 1-amino-9-
aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid (PEG18)) on the hydrophilicity of (5-(4-carboxyphenyl)-10,15, 20-triphenyl
chlorin coupled to DKPPR or TKPRR peptide via these spacers. The reverse phase retention times for TPC–Ahx–DKPPR, TPC–
PEG9–DKPPR and TPC–PEG18–DKPPR were 19.35, 19.05 and 18.02 min respectively. For TPC–Ahx–TKPRR, TPC– PEG9–
TKPRR and TPC– PEG18–TKPRR we found 17.37, 17.31, and 16.94 min. The retention time of the conjugate was inversely
proportionated to their polarity. By increasing the length of our spacer, we probably induced a decrease in the polarity of our
targeted photosensitizer. If we consider the parameter “hydrophobicity”, the best candidates could be Ce6 and PpIX.
Figure 2 shows the comparison of the hydrophobicities of PS and FA-targeted PS. Each FA-targeted PS presents several
retention times due to the formation of isomers. Indeed, carbodiimide-activated FA can link to either ꢀ-or γ-carboxyl groups of
the glutamate residue. For example, TPC possesses two isomers and then TPC-OEG-FA possesses four due to the coupling of
each TPC isomer to ꢀ- or γ−carboxyl groups of FA. By RP-HPLC, we determined that around 70% of the PSs were linked
through the γ-carboxyl group. This is important information since only the γ-conjugate can bind to FR.
new figure
Figure 2. Comparison of the hydrophobicities of PS and FA-targeted PS (reverse phase HPLC (C18 column),
acetonitrile/water gradient (10/90 % à 100/0 % in 25 min, 100/0% for 15 min, acetonitrile/water, v/v)). UV-Detection at 415 nm.
1.3. Photophysical properties
We recorded the absorption and fluorescence spectra of all the photosensitizers in DMSO. DMSO was chosen because it was
the only solvent in which all the photosensitizers were totally soluble. In figures 3 and 4, the absorption and fluorescence spectra
of the non-targeted and FA-targeted photosensitizers are included.

new figure
Figure 3. (a) Absorption spectra of photosensitizers in DMSO; (b) Absorption spectra of FA-targeted photosensitizers in
DMSO at the same concentration (C= 4.9 E-6 M)
new figure
Figure 4. (a) Fluorescence spectra of photosensitizers in DMSO; (b) Fluorescence spectra of FA-targeted photosensitizers in
DMSO at the same DO.
The photophysical properties of PSs do not change drastically after coupling with FA as seen in Table 2 where we reported
epsilons, fluorescence and singlet oxygen quantum yields and lifetimes.
The introduction of FA can lead to changes in the photophysical properties which are different for the various compounds.
Nevertheless, all of the FA targeted-PSs were still found to produce fluorescence and 1O2. For Ce6 a decrease of Φf and Φ∆ can
be observed. For TPC-COOH, Φ∆ decreases whereas Φf is not modified. For TPP-COOH and PpIX Φf were found to
decrease whereas
increased. It was found that TPP-FA is a good fluorescent probe and a good singlet oxygen photosensitizer
while TPP-NH2 is much less efficient.This phenomena might be due to the quenching of the singlet excited state by the amino
moietie due to a photoinduced electron transfer as suggested by Jiang et al.[44]
Table 2. Photophysical properties of FA-targeted photosensitizers in DMSO.
Compound
ꢁ
ꢂexc
(nm)
ꢄf (ns) ꢄꢃ(µs)
Φf Φꢃ
(%) (%)
(L.mol−1.cm−1)
Soret Band Soret Band
(ꢂmax)
QIV
QIII
QII
QI 285 nm
(420 nm)
411224
(418 nm)
TPP-COOH
TPP-OEG-FA
TPC-COOH
dTPC-OEG-FA
PpIX
391 429 17 347 8367 5510 4 898 13061 414 15 39 11.5 8.3
231224
(419 nm)
223 673 7755 2653 1020 1429 31837 414
9
51 11.4
6
77347
(420 nm)
77 347
4898 3469 1429 9796 4082 414 44 51 10.3 11.6
120816
(420 nm)
120 816 6735 5918 3265 21224 53877 414 46 24 11.1 10.1
165510
(406 nm)
93 469 15918 12857 8367 6122 12041 414
9
8
27 15.1 6.8
43 12.6 6.9
76531
(407 nm)
PpIX-OEG-FA
Ce6
47 347
5714 4286 2449 1224 37551 414
113218
(405 nm)
45 542 10759 5701 4761 35773 14732 414 31 53 5.9
7.1
8.2
0
119739
(406 nm)
Ce6-OEG-FA
TPP-NH2
53 796
8736 2299 1954 35251 39427 414 15 31 4.7
266531
(419 nm)
263 673 16939 12449 7347 6939 17959 414
1
0
1.3
11
351429
(420 nm)
TPP-FA
351 429 15918 9184 5510 5306 44490 414 10 63
6.4
Photodynamic dose is proportional to [PS]*(j*t)*[3O ]*ε*ꢅꢃ where [PS] is the PS concentration, (j*t) is the light fluence, [3O ]
2
2
= the concentration of O2 in the medium, ε = the molar extinction coefficient and ꢅꢃ the singlet oxygen quantum yield. In the
case of PDT treatment, it should be possible to keep (j*t), [3O2] and [PS] constant for all compounds. The efficiency will be due
to ε and ꢅꢃ.
In figure 5, we have described the Epsilon of QI, fluorescence quantum yield and singlet oxygen quantum yield of the FA-
targeted PSs in DMSO to determine which FA-targeted PS would be the best for PDT applications, that is to say which PS

presents the best epsilon and singlet oxygen quantum yield, as well as a modest fluorescence quantum yield enabling the
detection of the compound.
new figure
Figure 5. Epsilon of QI, fluorescence quantum yield and singlet oxygen quantum yield of the FA-targeted photosensitizers in
DMSO.
If we consider the parameters ꢅꢃ and epsilon at QI, Ce6 seems the best compound. Nevertheless, in clinical practice for
porphyrin and chlorin, two lasers sources are mainly used, 635 nm for PpIX and 652 nm for the chlorin Foscan. In table 3, we
report ε (635 nm)* ꢅꢃ and ε (635 nm)* ꢅꢃ for all the FA-targeted photosensitizers.
Table 3. ε (635 nm)*ꢅꢃ and ε (652 nm)*ꢅꢃ for all the FA-targeted photosensitizers.
compound
ε (635 nm)
2653
612
ε (652 nm)
408
ε (635 nm)*
1671
312
ε (652 nm)*
257
TPP-FA
0.63
0.51
0.24
0.31
0.43
TPP-OEG-FA
TPC-OEG-FA
Ce6-OEG-FA
PpIX-OEG-FA
204
104
2245
204
21224
14286
204
539
5094
4429
88
63
408
175
Clearly for an excitation at 635 nm, TPP-FA is the PS that absorbs the most. For an excitation at 652 nm, TPC-OEG-FA and
Ce6-OEG-FA present the highest ε (652 nm)*ꢅꢃ. Moreover, these two compounds are the most hydrophilic.
Finally, if we take into account the water solubility and all the photophysical parameters, Ce6-OEG-FA appears to be
the best candidate. This is not surprising and was indeed submitted by K.S. Park in a patent in 2014[35]. However the synthesis
of this compound is not a trivial matter because of its three free carboxylic functions. Some isomers can be formed during
synthesis and purification of the required isomer can be time-consuming.
2. Conclusion
We synthesized 5 FA-targeted photosensitizers. We choose two different classes of photosensitizers - “synthetic”
photosensitizers i.e. a porphyrin, a chlorin, and “natural” photosensitizers i.e PpIX and Ce6. An OEG spacer was added between
the FA and the photosensitizer except for one compound (a porphyrin directly linked to FA). We can conclude that for these
photosensitizers, the introduction of an OEG does not significantly improve the hydrophilicity of the FA-porphyrin. Moreover,
all the FA-targeted photosensitizers present good to very good photophysical properties. The best one appears to be Ce6 even if
this compound might be difficult to synthesize in large quantities and with a high level of purity due to its chemical structure. In
order to evaluate the influence on the photosensitizers onto affinity for FA receptor, we plan to perform affinity experiments in
vitro on cells expressing RAF (KB) and cells that do not express RAF as well as competition assays. For the best candidate, in
vivo tests will be also performed.
3. Experimental section
3.1. Chemicals
Chlorin e6 (Ce6 4) was purchased from Frontier Scientific. 5-(4-aminophenyl)-10,15,20-triphenylporphyrin (TPP-NH2 5) was
purchased from Porphychem. Protoporphyrin IX (PpIX 3), N-Boc-2,2´-(ethylenedioxy)diethylamine (MWOEG-Boc = 248.32 g/mol)
and all other chemicals were obtained from Sigma-Aldrich. Reverse-phase high-performance liquid chromatography (HPLC)
was performed on Prostar HPLC (Varian). Analytical HPLC were done with a Pursuit 5-C18 column (2.5 µm, 4.6 × 150 mm,
Varian) and preparative HPLC with Pursuit 5-C18 column (5 µm, 21.2 × 150 mm, Varian), both using a photodiode array detector
(UV-Visible detection, Varian) and a fluorescence detector (Varian). NMR spectra (1H, COSY and TOCSY) were recorded on a
BRUKER AVANCE spectrometer at 300 MHz. Mass spectra were recorded on LCMS-2010 EV of Shimazu. High resolution
mass spectrometry (HRMS) experiments were performed on a micro-Tof Bruker (electrospray ionization ESI +, 50-1000 in low
and 50-2500 in width).
Absorption spectra were recorded on a UV-3600 UV-visible double beam spectrophotometer (SHIMADZU, MARNE LA
VALLEE, France). Fluorescence spectra were recorded on a Fluorolog FL3-222 spectrofluorimeter (HORIBA Jobin Yvon,
LONGJUMEAU, France) equipped with 450 W Xenon lamp, a thermo-stated cell compartment (25°C), a UV-visible
photomultiplier R928 (HAMAMATSU Japon) and an InGaAs infrared detector (DSS-16A020L Electro-Optical System Inc,
Phoenixville, PA, USA).

Excitation beam is diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 330 nm). Emission
beam is diffracted by a double ruled grating SPEX monochromator (1200 grooves/mm blazed at 500 nm). Singlet oxygen
emission was detected through a double ruled grating SPEX monochromator (600 grooves/mm blazed at 1 µm) and a long-wave
pass (780 nm). All spectra were measured in 4 faces quartz cuves. All the emission spectra (fluorescence and singlet oxygen
luminescence) have been displayed with the same absorbance (less than 0.2) with the lamp and photomultiplier correction.
Time-resolved experiments were performed using for excitation: a pulsed laser diode emitting at 407 nm (LDH-P-C-400M,
FWHM < 70 ps, 1 MHz) coupled with a driver PDL 800-D (both PicoQuant GmbH, BERLIN, Germany) and for detection: an
avalanche photodiode SPCM-AQR-15 (EG & G, VAUDREUIL, Canada) coupled with a 650 nm long-wave pass filter as
detection system. The acquisition was performed by a PicoHarp 300 module with a 4 channels router PHR-800 (both PicoQuant
GmbH, BERLIN, Germany). The fluorescence decays were recorded using the single photon counting method. Data were
collected up to 1000 counts accumulated in the maximum channel and analyzed using Time Correlated Single Photon Counting
(TCSPC) software Fluofit (PicoQuant GmbH, BERLIN, Germany) based on iterative reconvolution using a Levensberg-
Marquandt algoritm, enabling the obtention of multi-exponential profiles (mainly one or two exponentials in our cases).
Singlet oxygen lifetime measurements have be performed on a TEMPRO-01 spectrophotometer (HORIBA Jobin Yvon,
LONGJUMEAU, France) composed with a pulsed diode excitation source SpectraLED-415 emitting at 415 nm, a cell
compartment, a Seya-Namioka type emission monochromator (600 – 2000 nm) and a H10330-45 near-infrared photomultiplier
tube with thermoelectric cooler (HAMAMATSU, Japan) as detection system. The system was monitored by a single photon
counting controller FluoroHub-B and the software DataStation and DAS6 (HORIBA Jobin Yvon).
3.2. Synthesis of Photosensitizers
3.2.1. Synthesis of 5-(4-carboxyphenyl)–10,15,20–triphenylporphyrin (TPP-COOH) 1
3.5 mL of freshly distilled pyrrole (50 mmol), 3.8 mL of benzaldehyde (37.5 mmol) and 4-carboxybenzaldehyde (1.875 g,
12.5 mmol) were poured into degassed CH2Cl2 (700 mL). After 15 min, 0.615 mL of boron trifluoride diethyl ether (5 mmol)
was added and the reaction was stirred for 2 hours. Then, p-chloranil (9.225 g, 37.5 mmol) was introduced. The mixture was
stirred at 60 °C overnight in the dark, under nitrogen. The solvent was removed. Column chromatography on silica gel
(CH2Cl2/EtOH, 97/3, v/v) was achieved to purify the crude product, yielding compound 1 (738.5 mg, 11%) as purple crystals. Rf
= 0.35 (CH2Cl2/EtOH = 97:3, v/v). 1H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -2.92 (s, 2H, NHpyrrole), 7.83 (s, 9H, Hm- and p-phenyl),
8.24 (d, 6H, Ho-phenyl), 8.37 (dd, 4H, Ho-phenyl-COOH), 8.84 (s, 8H, Hꢇ-pyrrole), 13.24 (br, 1H, COOH). HRMS (ESI+): m/z
calculated for C45H30N4O2 [M+H]+ 659.2442; found 659.2476.
3.2.2. Synthesis of 5-(4-carboxyphenyl)-10,15,20-triphenyl chlorin (TPC-COOH) 2
The synthesis was performed according to the protocol described by Laville et al.[45]: 1 (200 mg, 0.30 mmol), K2CO3 (1.26 g;
9 mmol) and p-toluenesulfonylhydrazide (1.70 g, 9 mmol) were dissolved in freshly distilled pyridine (20 mL) and stirred
overnight at pyridine reflux (110 °C) in a round bottom flask isolated from light and under inert atmosphere. After cooling, ethyl
acetate (50 mL) and water (50 mL) were added and the solution was heated at 100 °C for 1 hour. The organic phase was
separated and washed with HCl (1 M, 200 mL), water (500 mL) and a saturated water solution of NaHCO3. By UV–visible
spectroscopy we could control the presence or not of chlorin and bacteriochlorin (bands at 650 and 735 nm, respectively). Then
the organic phase was concentrated and lyophilized. The dark red solid obtained was dissolved in ethyl acetate (50 mL).
Additions of 100 µL of o-chloranil (o-chloranil solution 300 mg/mL in EtOAc) were poured every 30 min into solution under
stirring at room temperature until the disappearance of the 735 nm-bacteriochlorin absorption peak. 100 mL of water was added
to quench the reaction (o-chloranil in excess). The solution was washed with an aqueous solution of NaHSO3 (5%, 200 mL) in
500 mL water and the organic phase was concentrated. The crude product was purified by HPLC using a MeOH/water (0.1%
TFA) [75:25] to MeOH 100% gradient in 30 min, followed by 15 min of isocratic MeOH. Rt = 15.6 and 16.6 min. Yield in 2
1
(169.9 mg, 61%). HNMR (300 MHz, DMSO-d6): ꢆ (ppm) = -1.59, -1.52 (2 x s, 2H, NHpyrrole), 4.13 (s, 4H, CH2), 7.74 (s, 9H,
H
m- and p-phenyl), 7.91, 8.06 (2 x d, 2H + 4H, Ho-phenyl), 8.18 (dd, 4H, Ho-phenyl-COOH), 8.30 (s, 4H, Hꢇ-pyrrole), 8.59 (s, 2H, Hꢇ-pyrrole).
HRMS (ESI+): m/z calcd. for C45H32N4O2 [M+H]+ 661.2598; found 661.2636.
3.3. Synthesis of succinimide ester Photosensitizers 6, 7
In the dark and under an inert atmosphere, the corresponding PS-COOH (1 eq.) was dissolved in CH2Cl2 (20-50 mL) and N-
hydroxysuccinimide (3 eq.) and N,N'-dicyclohexylcarbodiimide (3 eq.) were added. The mixture was stirred at 40 °C, for 4
hours, under argon.
3.3.1. Synthesis of 5-(4-carboxyphenylsuccinimide ester)–10,15,20–triphenylporphyrin (TPP-NHS) 6
Purification of the crude material was performed using a silica gel column with CH2Cl2. Compound 6 was obtained after
recrystallization in CH2Cl2/hexane, as a purple solid at a yield of 75% (377.3 mg). Rf = 0.79 (CH2Cl2/EtOH = 97/3, v/v). H
1
NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -2.91 (s, 2H, NHpyrrole), 3.00 (s, 4H, CH2), 7.84 (s, 9H, Hm- and p-phenyl), 8.22 (d, 6H, Ho-
phenyl), 8.50 (dd, 4H, o-phenyl-COOH),), 8.85 (s, 8H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C49H33N5O4 [M+H]+ 756.2605; found
756.2588.
3.3.2. Synthesis of 5-(4-carboxyphenylsuccinimide ester)-10,15,20-triphenylchlorin (TPC-NHS) 7
Purification of the crude material was performed using a silica gel column with CH2Cl2. Compound 7 was obtained, after
recrystallization in CH2Cl2/hexane, as a purple solid with a yield of 89% (102.6 mg). Rf = 0.17 (CH2Cl2 = 100 %). 1HNMR (300
MHz, DMSO-d6): ꢆ (ppm) = -1.61, -1.48 (2 x s, 2H, NHpyrrole), 2.98 (s, 4H, NHS-CH2), 4.13 (s, 4H, CH2), 7.74 (s, 9H, Hm- and p-
phenyl), 7.90, 8.08(2 x d, 2H + 4H, Ho-phenyl), 8.19 (dd, 4H, Ho-phenyl-COOH), 8.32 (s, 4H, Hꢇ-pyrrole), 8.42 (s, 2H, Hꢇ-pyrrole). HRMS
(ESI+): m/z calcd. for C49H35N5O4 [M+H]+ 758.2762; found 758.2796.

Following procedures were adapted from previous protocols.[27, 46]
3.4. Synthesis of Photosensitizer-OEG-NHBoc derivatives 8, 9, 15 and 16
3.4.1. Synthesis of TPP-OEG-NHBoc 8
84 mg of compound 6 (0.11 mmol) and 26.3 mg of N-Boc-2,2´-(ethylenedioxy)diethylamine (0.11 mmol) were dissolved in 30
mL of THF. The reaction mixture was stirred at room temperature, under a nitrogen atmosphere in the dark for 18 hours. The
solvent was removed by vacuum. Silica gel column with 3% EtOH in CH2Cl2 was used to purify the crude material and
compound 8 was obtained as a purple solid with a yield of 73% (68.7 mg). Rf = 0.32 (CH2Cl2/EtOH = 97/3, v/v). 1H NMR (300
MHz, DMSO-d6): ꢆ (ppm) = -2.92 (s, 2H, NHpyrrole), 1.35 (s, 9H, Boc), 3.11 (q, 2H, CH2 OEG), 3.45 (q, 2H, CH2 OEG), 3.60 (m, 8H,
CH2 OEG), 7.81 (s, 9H, Hm- and p-phenyl), 8.22 (d + s, 10H, Ho-phenyl-COOH and Ho-phenyl), 8.83 (s, 8H, Hꢇ-pyrrole). HRMS (ESI+): m/z
calcd. for C56H52N6O5 [M+H]+ 889.4072; found 889.4059.
3.4.2. Synthesis of TPC-OEG-NHBoc 9
114 mg of compound 7 (0.15 mmol) and 37.6 mg of N-Boc-2,2´-(ethylenedioxy)diethylamine (0.15 mmol) were dissolved in
30 mL of THF. The reaction mixture was stirred at room temperature under nitrogen in the dark for 24 hours. The solvent was
removed under vacuum. Silica gel column with 3% EtOH in CH2Cl2 was used to purify the crude material and compound 9 was
1
obtained as a purple solid with a yield of 78% (105.3 mg). Rf = 0.34 (CH2Cl2/EtOH = 97/3, v/v). H NMR (300 MHz, DMSO-
d6): ꢆ (ppm) = -1.58, -1.53 (2 x s, 2H, NHpyrrole), 1.35 (s, 9H, Boc), 3.09 (q, 2H, CH2 OEG), 3.42 (q, 2H, CH2 OEG), 3.61 (m, 8H,
CH2 OEG), 4.13 (s, 4H, CH2), 7.73 (s, 9H, Hm- and p-phenyl), 7.92, 8.06 (2 x d, 2H + 4H, Ho-phenyl), 8.10 (dd, 4H, Ho-phenyl-COOH), 8.27 (s,
4H, Hꢇ-pyrrole), 8.57 (s, 2H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C56H54N6O5 [M+H]+ 891.4228; found 891.4214.
3.4.3. Synthesis of PpIX-OEG-NHBoc 15
N-Boc-2,2´-(ethylenedioxy)diethylamine (1.1 eq.) was dissolved in 2 mL of DMF. Protoporphyrin IX (100 mg) and N,N-
dicyclohexylcarbodiimide (DCC) (1.1 eq.) dissolved in dry DMF (5 mL) were added. 1-hydroxybenzotriazole (HOBt) (1.1 eq.)
was then introduced. The mixture protected from light was stirred for 24 hours under argon. DMF was then evaporated and the
crude product was solubilized in CH2Cl2. The organic layer was washed with water (2 x 50 mL), dried with MgSO4, and CH2Cl2
1
was evaporated. The pure product 15 was obtained with a yield of 43% after purification with silica gel column. H NMR (300
MHz, CDCl3): ꢆ (ppm) = 1.28 (s, 9H, Boc), 1.87 (m, 2H, CH2), 2.23 (m, 2H, CH2), 2.48 (m, 2H, CH2), 2.73 (m, 2H, CH2), 3.52 -
2.90 (m, 20H, 4CH3ꢇ pyrrole + 4CH2 OEG), 4.02 (brd, 2H, CH2), 4.18 (brd, 2H, CH2), 4.53 (brs, 1H, NHCO), , 6.31 - 6.05 (m, 4H,
CH2vinyl), 6.64 (s, 1H, NHBoc), 8.17 – 7.93 (m, 2H, CHvinyl), 9.41, 9.64, 9.80 (s, 4H, Hmeso). MS (ESI+): m/z calcd. for
C45H56N6O7 [M+H]+ 792.96; found 792.9603.
3.4.4. Synthesis of Ce6-OEG-NHBoc 16
N-Boc-2,2´-(ethylenedioxy)diethylamine (1 eq.) was dissolved in 1 mL of DMF. A solution of chlorin e6 4 (200 mg) and N,N-
dicyclohexylcarbodiimide (DCC) (1 eq.) in dry THF/DMF (25/2.5 mL) was added. 1-hydroxybenzotriazole (HOBt) (1.1 eq.) was
then introduced. The mixture protected from light was stirred for 24 hours under argon. Solvents were evaporated under vacuum.
1
After purification by column chromatography, the pure product 16 was obtained with a yield of 39% (109 mg). H NMR (300
MHz, DMSO-d6): ꢆ (ppm) = -2.65 (s, 1H, NHpyrrole), -2.03 (s, 1H, NHpyrrole), 1.23 (s, 3H, CH3), 1.27 (m, 9H, Boc), 1.37 (s, 3H,
CH3ꢇ pyrroline), 1.69 (m, 8H, CH2), 2.84 (m, 2H, CH2), 3.06 (m, 6H, CH2), 3.43 (s, 3H, CH3ꢇ pyrrole), 3.48 (s, 3H, CH3ꢇ pyrrole), 3.54
(s, 3H, CH3ꢇ pyrrole), 3.82 (brd, 2H, CH2-CH3), 4.33 (brs, 1H, Hꢇ pyrroline), 4.53 (brd, 1H, Hꢇ pyrroline), 5.10 (m, 2H, CH2-CO), 5.81
(m, 1H, NHCO), 6.15 (d, 1H, CH2vinyl), 6.44 (d, 1H, CH2vinyl), 8.35 (dd, 1H, CHvinyl), 9.13 (s, 1H, Hmeso), 9.66 (s, 1H, Hmeso), 9.78
(s, 1H, Hmeso). MS (ESI+): m/z calcd. for C45H56N6O7 [M+H]+ 826.96; found 826.9663
3.5. Synthesis of PS-OEG-NH2 derivatives 10, 11, 17 and 18
PS-OEG-NHBoc molecules were dissolved in 2 mL of TFA. The mixture protected from light was stirred for 2 hours under
argon. Next, TFA was lyophilized. The colored residue was solubilized in CH2Cl2 (10 mL), and anhydrous potassium carbonate
was added until the color changed from green to red (for TPP-COOH and TPC-COOH) or from blue to green (for Ce6). After
filtration, solvent was evaporated.
3.5.1. Synthesis of TPP-OEG-NH2 10
Silica gel column with CH2Cl2/MeOH (from 80:20 to 50:50, v/v) was used to purify the crude compound yielding 10 (75.1
1
mg, 85%) as a purple solid. Rf = 0.72 (CH2Cl2/MeOH = 1/1, v/v). H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -2.92 (s, 2H,
NHpyrrole), 3.33 (m, 2H, CH2 OEG), 3.64 (m + d, 10H, CH2 OEG), 7.83 (s, 9H, Hm- and p-phenyl), 8.22 (d + s, 10H, Ho-phenyl-COOH and Ho-
phenyl), 8.84 (s, 8H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C51H44N6O3 [M+H]+ 789.3548; found 789.3560.
3.5.2. Synthesis of TPC-OEG-NH2 11
The residue was purified by HPLC on a C18 preparative column using a MeOH/water gradient (75/25 % to 100/0 % in 30 min,
100/0% for 15 min, MeOH/water (0.1% TFA), v/v). The fraction Rt = 8.05 and 9.54 min gave the pure product 11 (21.0 mg,
24%). 1H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -1.56, -1.51 (2 x s, 2H, NHpyrrole), 3.02 (q, 2H, CH2 OEG), 3.58 (q, 2H, CH2 OEG),
3.74 (s, 8H, CH2 OEG), 4.11 (s, 4H, CH2), 7.70 (s, 9H, Hm- and p-phenyl), 8.00, 8.07 (2 x d, 2H + 4H, Ho-phenyl), 8.20 (dd, 4H, Ho-phenyl-
COOH), 8.32 (s, 4H, Hꢇ-pyrrole), 8.57 (s, 2H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C51H46N6O3 [M+2H]2+ 396.1880; found
396.1889.
3.5.3. Synthesis of PpIX-OEG-NH2 17
Compound 15 was dissolved in 2 TFA (2mL) and let under stirring for 2 hours under nitrogen and protected by light. Next,
TFA was lyophilized. The red residue was solubilized in CH2Cl2/EtOH (20 mL), and anhydrous potassium carbonate was added
until the color changed. After filtration, solvent was evaporated. HPLC on a C18 preparative column with a acetonitrile/water

gradient (10/90 % to 100/0 % in 45 min, 100/0% for 5 min, acetonitrile/water, v/v) was used to purify the residue. The fraction Rt
1
= 21.7 min gave the pure product 17 (25 mg, 49%). H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = 2.67 (brq, 2H, CH2), 2.79 (m,
4H, CH2), 2.80 – 3.09 (m, 8H, CH2 OEG), 3.20 (brt, 2H, CH2 OEG), 3.57 (brd, 6H, CH3 ꢇ-pyrrole), 3.63 (bt, 6H, CH3ꢇ-pyrrole), 4.30 (brd,
4H, CH2), 6.19 (brd, 2H, CH2 vinyl), 6.38 (brd, 2H, CH2vinyl), 7.58 (brs, 2H, NH2), 7.82 (brt, 1H, NHCO), 8.45 – 8.35 (m, 2H, CH
vinyl), 10.11 – 10.03 (m, 4H, H meso). MS (ESI+): m/z calcd. for C45H56N6O7 [M+H]+ 692.85; found 692.8527
3.5.4. Synthesis of Ce6-OEG-NH2 18
Compound 16 (109 mg) was dissolved in TFA (4 mL) and let under stirring for 2 hours under nitrogen and protected by light.
Next, TFA was lyophilized. The crude product was solubilized in CHCl3/EtOH (2/1), and anhydrous potassium carbonate was
added until the color changed from purple to green. After filtration, the solvent was evaporated. HPLC with a C18 preparative
column and an acetonitrile/water gradient (10/90 % to 100/0 % in 45 min, 100/0% for 5 min, acetonitrile/water, v/v) was used to
1
purify the compound. The fraction Rt = 22.7 min gave the pure product 18 (50 mg, 52%). H NMR (300 MHz, DMSO-d6): ꢆ
(ppm) = -2.68 (s, 1H, NHpyrrole), -2.03 (s, 1H, NHpyrrole), 1.23 (s, 3H, CH3), 1.35 (s, 3H, CH3ꢇ pyrroline), 1.68 (m, 8H, CH2), 2.83 (m,
2H, CH2), 3.04 (m, 6H, -CH2), 3.41 (s, 3H, CH3ꢇ pyrrole), 3.46 (s, 3H, CH3ꢇ pyrrole), 3.52 (s, 3H, CH3ꢇ pyrrole), 3.81 (brd, 2H, CH2),
4.31 (brs, 1H, Hꢇ pyrrole), 4.51 (brd, 1H, Hꢇ pyrrole), 5.73 (m, 2H, CH2-CO), 6.13 (d, 1H, CH2vinyl), 6.43 (d, 1H, CH2vinyl), 7.58 (m,
2H, NH2), 8.01 (brs, 1H, CONH), 8.35 (dd, 1H, CHvinyl), 9.11 (s, 1H, Hmeso), 9.62 (s, 1H, Hmeso), 9.77 (s, 1H, Hmeso). MS (ESI+):
m/z calcd. for C40H50N6O7 [M+H]+ 726.86; found 726.8579
3.6. Synthesis of Photosensitizer-Spacer-FA derivatives 12, 13, 19 and 20
FA (1 eq.) and DCC (1 eq.) were dissolved in anhydrous DMSO (5 mL) and pyridine (2 mL). The mixture was stirred for 15
min protected by light and under a N2 atmosphere. Compound PS-OEG-NH2 (0.9 eq.) was then added and the stirring was
continued for 24h. The solution was slowly poured into vigorously stirred cold diethyl ether. By centrifugation, the precipitate
obtained was collected and washed with diethyl ether. The powder was dried under high vacuum. HPLC on a C18 preparative
column with an acetonitrile/water gradient (10/90 % to 100/0 % in 25 min, 100/0% for 15 min, acetonitrile/water, v/v) was used
to purify the compound.
3.6.1. Synthesis of TPP-OEG-FA 12
The fraction Rt = 17.9 and 18.0 min gave the pure product 12 (30 mg, 28%). 1H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -2.92
(s, 2H, NHpyrrole), 2.07 (m, 2H, CH2 FA), 2.30 (m, 2H, CH2 FA), 3.60 (m, 8H, CH2 OEG), 4.40 (m, 1H, NH FA), 4.48 (d, 2H, CH2 FA),
6.58 (d, 2H, Harom FA.), 6.88 (m, 3H, NH + NH2), 7.84 (s, 9H, Hm- and p-phenyl), 8.22 (d, 6H, Ho-phenyl), 8.31 (s, 4H, Ho-phenyl-COOH),
8.64 (d, 1H, CH arom FA), 8.84 (s, 8H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C70H61N13O8 [M+H]+ 1212.4839; found 1212.4790.
3.6.2. Synthesis of TPC-OEG-FA 13
No further purification by HPLC was necessary in this case. The fraction Rt = 20.8; 21.0; 21.2 and 21.4 min gave the pure
1
product 13 (21 mg, 62%). H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -1.59, -1.54 (2 x s, 2H, NHpyrrole), 2.00 (m, 2H, CH2 FA),
2.30 (t, 2H, CH2 FA), 3.57 (m, 8H, CH2 OEG), 4.12 (s, 4H, CH2), 4.3 (m, 1H, NH), 4.47 (d, 2H, CH2 FA), 6.65 (d, 2H, Harom FA), 6.91
(m, 3H, NH + NH2), 7.70 (s, 9H, Hm- and p-phenyl), 8.00, 8.07 (2 x d, 2H + 4H, Ho-phenyl), 8.11 (d, 1H, NH), 8.20 (dd, 4H, Ho-phenyl-
COOH), 8.30 (s, 4H, Hꢇ-pyrrole), 8.59 (s, 2H, Hꢇ-pyrrole), 8.64 (d, 1H, CH arom FA), 11.40 (s, 1H, OH). HRMS (ESI+): m/z calcd. for
C70H63N13O8 [M+H]+1214.4995; found 1214.4968.
3.6.2.1. Synthesis of PpIX-OEG-FA 19
In the dark and under a N2 atmosphere, FA (1 eq.), DCC (1 eq.) and NHS (1 eq.) were dissolved in anhydrous DMSO (2 mL) .
The solution was stirred overnight. FA-NHS was added dropwise to compound 17 (1 eq.) dissolved in 1 mL of DMSO and 0.5
mL of pyridine the stirring was continued for 4 days. The solution was slowly poured into vigorously stirred cold diethyl ether.
The precipitate was obtained by centrifugation, washed with both diethyl ether and CH2Cl2. The purple powder was dried and 19
was obtained with a yield of 53% (20 mg). 1H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -3.84 (s, 2H, NHpyrrole), 2.08 (m, 2H, CH2
FA), 2.29 (m, 2H, CH2 FA), 2.88 - 3.24 (m, 16H, CH2 + CH2 OEG), 3.63 (brd, 6H, CH3ꢇ pyrrole), 3.75 (brd, 6H, CH3ꢇpyrrol), 4.34 (brd,
4H, CH2), 4.48 (m, 3H, CH2 and CHCO FA), 6.20 (brd, 2H, CH2vinyl), 6.44 (brd, 2H, CH2vinyl), 6.55 (d, 2H, Harom FA), 6.86 (m, 3H,
NH + NH2), 7.63 (d, 2H, Harom FA), 7.82 (brt, 1H, NHCO), 8.11 (m, 1H, NH), 8.54 (m, 3H, CHvinyl, NHCO), 8.64 (s, 1H, Harom
FA), 10.27 – 10.35 (m, 4H, Hmeso), 11.39 (s, 1H, OH), 12.24 (brs, 2H, COOH). HRMS (ESI+): m/z calcd. for C59H65N13O10
[M+H]+ 1116.5; found 1116.5004.
3.6.3. Synthesis of Ce6-OEG-FA 20
In the dark and under a N2 atmosphere, FA (1 eq.), DCC (1 eq.) and NHS (1 eq.) were dissolved in anhydrous DMSO (2 mL),
the stirring was continued overnight. FA-NHS was added dropwise to compound 18 (1 eq., 30 mg) in 1 mL of DMSO and 0.5
mL of pyridine. The stirring was continued for 5 days. The solution was slowly poured into vigorously stirred cold diethyl ether.
The precipitate was obtained by centrifugation, washed with both diethyl ether and CH2Cl2. The green powder 20 was dried and
1
obtained with a yield of 45 % (36 mg). H NMR (300 MHz, DMSO-d6): ꢆ (ppm) = -2.12 (s, 1H, NH pyrrole), -1.74 (s, 1H, NH
pyrrole), 1.26 – 1.39 (m, 6H, CH3 and CH3ꢇ pyrroline), 1.76 (m, 2H, CH2), 2.05 (m, 2H, CH2 FA), 2.35 (m, 2H, CH2 FA), 2.87 - 3.28 (m,
16H, CH2 and CH2 OEG), 3.54-3.81 (m, 11H, CH2 and 3CH3ꢇ pyrroline ), 4.48 (m, 4H, CH2 and Hꢇ pyrroline), 5.17 - 5.39 (m, 2H), 6.17 –
6.90 (m, 6H, CH2 vinyl and 2 Harom FA), 7.65 – 8.30 (m, 5H), 8.64 (s, 2H), 9.10 (s, 1H, Hmeso), 9.74(s, 2H, Hmeso), 11.44 (s, 1H, OH),
12.2 (brs, 2H, COOH). HRMS (ESI+): m/z calcd. for C59H67N13O2 [M+H]+ 1150.5010; found 1150.6350.
3.7. Synthesis of direct linked TPP-FA 14
In the dark and under a N2 atmosphere, FA (74 mg; 0.17 mmol) and DCC (34 mg; 0.17 mmol) were dissolved in a mixture
anhydrous DMSO/pyridine (5 mL/2 mL). The solution was stirred for 15 min at room temperature. TPP-NH2 5 (105 mg; 0.17
mmol) was then added and the stirring was continued for 48h. The solution was slowly poured into vigorously stirred cold

diethyl ether. The precipitate was obtained by centrifugation, washed with diethyl ether, dried under high vacuum and purified by
HPLC with a C18 preparative column using an acetonitrile/water gradient (10/90 % to 100/0 % in 45 min, 100/0% for 15 min,
acetonitrile/water, v/v). The fraction Rt = 27.4 and 28.3 min gave the pure product 14 (7.3 mg, 4%). 1H NMR (300 MHz, DMSO-
d6): -2.90 (s, 2H, NH pyrrole), 2.00 (m, 2H, CH2 FA), 2.35 (t, 2H, CH2 FA), 4.53 (m, 3H, NH + CH2), 6.67 (dd, 2H, Harom), 7.48 (br,
2H, NH2), 7.65 (d, 2H, CHarom), 7.80 (d, 2H, CHarom.), 7.83 (s, 9H, Hm- and p-phenyl), 8.05 (d, 1H, NH.), 8.15 (dd, 4H, Ho-phenyl-COOH),
8.28 (d, 6H, Ho-phenyl), 8.72 (d, 1H, CHarom), 8.84 (s, 8H, Hꢇ-pyrrole). HRMS (ESI+): m/z calcd. for C63H48N12O5 [M+Na]+
1075.3763; found 1075.3741.
3.8. Photophysical Properties
The protocols for absorption, fluorescence, and lifetime have been already described elsewhere.[47]
The quantum yield of fluorescence is determined thanks to the equation (1):
ꢃ
ꢆꢇ
ꢈ
ꢅ
Φꢀ = Φꢀꢁꢂ. ꢄ ꢂ. ꢆꢇ ꢂ. (ꢈ )²
(1)
ꢃ
ꢅ
ꢄꢅ
where ꢅf and ꢅf0, If and If0, DO and DO0, n and n0 are the fluorescence quantum yields, the fluorescence intensities, the
optical densities at the excitation wavelength and the refraction indices of the sample and of the reference, respectively.[48, 49]
The quantum yield of singlet oxygen production is determined thanks to the equation (2):
ꢃ
Φꢉ = Φꢉꢁꢂ. ꢂ. ꢆꢆꢇꢇ ꢂ
(2)
ꢅ
ꢃ
ꢊꢅ
where ꢅꢃ and ꢅꢃ0, Iꢃ and Iꢃ0, DO and DO0 are the luminescence quantum yields, the luminescence intensities and the optical
densities at the excitation wavelength of the sample and of the reference (Rose bengal ꢅꢃ0 = 0.16) respectively.[50]
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N
HN
N
NH
N
N
NH
N
NH
N
NH
N
N
N
N
O
O
NH2
NH
HN
OH
OH
HN
HN
HN
COOH
COOH
O
HOOC
OH
OH
O
TPP-COOH
PpIX
Ce6
TPC-COOH
TPP-NH2
1
3
5
2
4
no linker or
O
COOH
γ
Photosensitizer
(Porphyrin or Chlorin)
O
N α
H
H
N
O
O
N
N
O
N
H
HN
N
H
Folic acid
OEG
H2N
N
Figure 1. Carrier linked folic acid conjugate system.

Scheme 1. Synthesis strategy for PS-OEG-FA 12, 13. (i) NHS, DCC, CH2Cl2, 45 °C, overnight. (ii) OEG (N-
Boc-2,2´-(ethylenedioxy)diethylamine), THF, r.t., 18-24 h. (iii) TFA, r.t., 2h. (iv) FA, DCC, pyridine, DMSO,
r.t., 24h

H
N
NH
N
N
O
4 eq.
3 eq.
1 eq.
OH
HN
O
O
(ii)
NH
N
N
O
(i)
(iii)
OH
HN
(ii)
NH
N
O
TPP-COOH
1
OH
HN
N
HO
O
TPC-COOH
2
Scheme 2. Synthesis of photosensitizers TPP-COOH 1 and TPC-COOH 2. (i) BF3.(OEt)2, CH2Cl2, reflux,
overnight and then, p-chloranil. (ii) p-TSH, K2CO3, pyridine, reflux, overnight. (iii) EtOAc, o-chloranil.

NH2
N
N
N
N
OH
HN
NH
N
NH
N
N
N
(i)
NH2
NH
O
HN
HN
HN
O
O
HO
TPP-FA 14
TPP-NH2 5
Scheme 3. Synthesis of TTP-FA 14: (i) FA, DCC, pyridine, DMSO, r.t., 48h.

30
25
20
15
10
PS
PS-OEG-FA
0
5
PpIX
6
C4e6
TPP1-NH2
TPP2-COOH TPC3-COOH
Photosensitizer
Figure 2. Comparison of the hydrophobicities of PS and FA-targeted PS (reverse phase HPLC (C18 column),
acetonitrile/water gradient (10/90 % à 100/0 % in 25 min, 100/0% for 15 min, acetonitrile/water, v/v)). UV-
Detection at 415 nm.

2
1,5
1
a)
TPP-COOH
PpIX
TPP-NH2
TPC-COOH
Ce6
0,5
0
260
360
460
560
660
760
Wavelength (nm)
b)
1,5
TPP-OEG-FA
PpIX-OEG-FA
TPP-FA
1
TPC-OEG-FA
Ce6-OEG-FA
0,5
0
260
360
460
560
660
760
Wavelength (nm)
Figure 3. (a) Absorption spectra of photosensitizers in DMSO; (b) Absorption spectra of FA-targeted
photosensitizers in DMSO at the same concentration (C= 4.9 E-6 M)

120000000
100000000
80000000
60000000
40000000
20000000
0
a)
TPP-COOH
PpIX
TPP-NH2
TPC-COOH
Ce6
600
620
640
660
680
700
720
740
760
780
800
Wavelength (nm)
b) 140000000
120000000
100000000
80000000
60000000
40000000
20000000
0
TPP-OEG-FA
PpIX-OEG-FA
TPP-FA
TPC-OEG-FA
Ce6-OEG-FA
600
620
640
660
680
700
720
740
760
780
800
Wavelength (nm)
Figure 4. (a) Fluorescence spectra of photosensitizers in DMSO; (b) Fluorescence spectra of FA-targeted
photosensitizers in DMSO at the same DO.

Fluorescence quantum yield
1O2 quantum yield
epsilon Q1
70
60
50
40
30
20
10
0
40
35
30
25
20
15
10
5
0
0
2
3
4
5
6
TPP1-FA
TPP-OEG-FA TPC-OEG-FA Ce6-OEG-FA PpIX-OEG-FA
PS-OEG-FA
Figure 5. Epsilon of QI, fluorescence quantum yield and singlet oxygen quantum yield of the FA-targeted
photosensitizers in DMSO.