Synthesis of porphyrin glycoconjugates bearing thiourea, thiocarbamate and carbamate connecting groups: Influence of the linker on chemical and photophysical properties

Article abstract of DOI:10.1016/j.dyepig.2014.03.029

In the present work, we have synthesized new porphyrin conjugates bearing thiourea, thiocarbamate and carbamate linkers using conventional and ultrasound-assisted methods. These molecules were synthesized by addition reactions using isothiocyanate derivat

Full text of DOI:10.1016/j.dyepig.2014.03.029

Dyes and Pigments 107 (2014) 69e80
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis of porphyrin glycoconjugates bearing thiourea,
thiocarbamate and carbamate connecting groups: Influence of the
linker on chemical and photophysical properties
a
b
a
b
Juliana C.C. Dallagnol , Diogo R.B. Ducatti , Sandra M.W. Barreira , Miguel D. Noseda ,
b
a,
*
Maria Eugênia R. Duarte , Alan Guilherme Gonçalves
Departamento de Farmácia, Universidade Federal do Paraná, Av. Lothario Meissner, 3400, Jardim Botânico, Curitiba, Paraná, Brazil
a
b
Departamento de Bioquímica, Universidade Federal da Paraná, Av. Cel. Francisco H. dos Santos, 100, Jardim das Américas, Curitiba, Paraná, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 January 2014
Received in revised form
In the present work, we have synthesized new porphyrin conjugates bearing thiourea, thiocarbamate
and carbamate linkers using conventional and ultrasound-assisted methods. These molecules were
synthesized by addition reactions using isothiocyanate derivates and amino- or hydroxyl-functionalized
meso-tetra(aryl)porphyrins to give porphyrin analogs linked to glycosyl or benzyl moieties. Glyco-
porphyrin derivates could be obtained with better yields than their respective benzyl analogs. Thiourea
and carbamate glycoporphyrins were also successfully synthesized through an ultrasound protocol with
a remarkable decreasing of the reaction time. By NMR experiments we were able to evaluated the linker
conformations which showed a preferred Z,Z conformation for the thiourea and carbamate derivatives. In
addition, porphyrins were evaluated in terms of their UVeVis spectra, singlet oxygen production, pho-
tostability and aggregation properties. Correlations between the linker type and the corresponding
photophysical properties were observed. Carbamate porphyrins seem to be the most promising photo-
sensitizers among the studied molecules due to their high singlet oxygen production, photostability and
absence of self-assembling behavior in aqueous media.
19 March 2014
Accepted 21 March 2014
Available online 29 March 2014
Keywords:
Porphyrins
Glycoporphyrins
Photostability
Photodynamic therapy
Singlet oxygen
Aggregation
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
surrounding healthy tissue during PDT course. In this way, conju-
gation of PS to molecular moieties that can be selectively recog-
Since porphyrins have become part of the therapeutic arsenal
for cancer treatment through photodynamic therapy (PDT),
numerous studies have been dedicated to the synthesis of new and
more efficient macrocyclic photosensitizers (PS). The first genera-
tion of PS is represented by hematoporphyrin derivates (e.g. Pho-
nized by tumor cells have become of interest. Conjugated PS are
then referred as the third generation of PS and their study currently
represents an active research area [3,6].
A number of structural motifs based on biomolecules, such as
proteins [7], immunoglobulins [8], cholesterol [9] and carbohy-
drates [10,11], have been chemically associated to macrocyclic rings
in order to enhance the selectivity of PS uptake by the target cell. In
this field, PS conceived through the attachment of glycosyl moieties
to the porphyrin ring have proved to be promising candidates when
targeted systems are desired [12]. This is related to the fact that
cancer cells present higher amounts of lectins (carbohydrate
binding proteins) on their surfaces than do regular cells [13,14].
Many porphyrin glycoconjugates have already been synthesized
with ether [15,16], ester [17] or triazole [18,19] groups as linkers for
binding the porphyrin to carbohydrate moieties. Nevertheless,
these linkers could not be considered ideal because the deglyco-
sylation process [20] as well as decreasing of biological activity [21]
have already been reported as result of in vitro assays employing
Ò
tofrin ), which gave place to a second generation that presented
improved chemical homogeneity and radiation absorptivity at
higher wavelengths of the visible spectrum [1e3]. However, both
first and second generations of PS typically act as unspecific cyto-
toxic drugs. In this case, PDT selectivity towards the tumor tissue
depends on the spatial focusing of the light source, as well as on the
more active metabolism presented by malignant cells, which am-
plifies the photodamage promoted by the PS [4,5]. On the other
hand, the lack of PS intrinsic selectivity can compromise the
*
Corresponding author. Tel.: þ55 41 3360 4065.
E-mail addresses: alan.goncalves@pq.cnpq.br,halagg@hotmail.com (A.G. Gonçalves).
http://dx.doi.org/10.1016/j.dyepig.2014.03.029
143-7208/Ó 2014 Elsevier Ltd. All rights reserved.
0

70
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
those PS glycoconjugates. Thus, it is considered important to syn-
thesize PS conjugates having different kinds of linkers in order to
evaluate their chemical stability and their influence on photody-
namic properties.
solution as reference or (b) a Bruker LTQ Orbitrap operating in the
ꢀ
1
positive-ion mode with porphyrins at 10
mg mL in methanol
using Ultramark 1621 as reference. Optical rotation values were
obtained with a Jasco P-200 polarimeter equipped with a sodium
ꢀ
1
The formation of thiourea bridges is a well-known approach for
binding different types of molecules through convergent synthe-
sis. This kind of linkage was first utilized for the attachment of
fluorescent probes to sensitive substrates, which could be per-
formed under very mild conditions in the absence of catalysts [22].
Because of the versatility and robustness of this functional group,
thiourea bridges became a common linker for binding carriers to
biologically active molecules. However, there are only a few ex-
amples of the synthesis of porphyrins bearing thiourea linkers.
Clark and Boyle (1999) [23] synthesized thiourea-containing
porphyrins by using isothiocyanate-functionalized porphyrins
and a series of amino compounds, including amino acids, aliphatic
and aromatic amines as starting materials. Other related studies
made use of reactions employing amino porphyrins and isothio-
cyanate compounds [24]. Thiourea-related linkers, such as thio-
carbamate and carbamate groups, have also facile synthesis
methodologies; nevertheless they are poorly explored for the
synthesis of conjugated porphyrins. Although the thiocarbamate
linker can be promptly obtained from reactions of hydroxyl
compounds with isothiocyanate derivates, to the best of our
knowledge there is no example of this kind of linkage involving
porphyrin synthesis. This fact could be related to the previously
noted instability of the thiocarbamate bridge [25,26]. There are
two reported examples of carbamate-linked porphyrins where
hydroxy-porphyrins and isocyanate-functionalized compounds
were utilized as starting materials [27,28]. The above cited linkers
have never been previously used for functionalizing porphyrins
with carbohydrate moieties. Also, a comparative evaluation of the
influence of such linkers on porphyrin photophysical properties is
yet to be performed. PS photophysical evaluation is considered key
in order to understand, or even predict, porphyrin photodynamic
potentialities [29].
light source from 1% (g 100 mL ) porphyrin solutions in DMSO.
UVeVis spectra were recorded with a Shimadzu UV-1800 spec-
trometer using porphyrin concentration at 12 mmol L in chlo-
ꢀ
1
roform. Fluorescence emission spectra were recorded with an
RF-5301PC Shimadzu spectrofluorometer using porphyrin con-
centrations as indicated in Fig. 8 and chloroform as solvent.
Infrared (IR) experiments were performed with KBr in an FTIR
Bomen/MB analyzer. The light source used for the photophysical
assays was a Lumacare LC 122A with a halogen/quartz 250 W
ꢁ
ꢀ1
lamp. All compounds were stored at 5 C in a 1 mmol L DMSO
solution (stock solutions) before the photophysical assays. These
experiments were conducted at room temperature in a dark room
to avoid any impact of ambient light over the main light source.
Compounds 9 and 11 were analyzed just after purification with
less than 24 h storing.
2.2. Synthesis and purification
Reagents and solvents were of reagent grade. For anhydrous
reactions, the solvents were dried according to stated methods [30].
Acetonitrile was dried by soaking it with silica gel for 24 h followed
by distillation. DMF was dried by soaking it for 24 h with anhydrous
4
MgSO . The dried solvents were then stored in flasks containing
ꢀ
molecular sieves 4 A under nitrogen atmosphere. Column chro-
matography was performed with the indicated solvents using
Sigma silica gel 60 (particle size 220e440 mesh). Yields refer to
chromatographically and spectroscopically pure compounds.
Compounds 1 [31], 2 [32], 3 [31], 4 [33], 5 [34] and 6 [35] are known
compounds and their spectroscopic data coincided to those re-
ported previously (for spectroscopy data see Supplementary
Information).
0 0 0 0
2.2.1. Synthesis of 5-[4-((2 ,3 ,4 ,6 -tetra-O-acetyl-b-D-
glucopyranosyl)thioureido)-phenyl]-10,15,20-triphenylporphyrin(7)
Method A: 5-(4-aminophenyl)-10,15,20-triphenylporphyrin 3
Here, we have investigated the synthesis of porphyrin conju-
gates bearing thiourea, thiocarbamate and carbamate linkers. For
this purpose, we utilized amino- or hydroxy-functionalized meso-
tetra(aryl)porphyrins and per-O-acetyl-
b
-D-glucopyranosyl iso-
(63 mg 0.1 mmol) and per-O-acetyl-b-D-glucosyl isothiocyanate 5
thiocyanate as starting materials. We have also employed benzyl
isothiocyanate in order to obtain non-glycosyl porphyrin conjugate
analogs for intercomparison of their properties. The linker confor-
mation of the porphyrin conjugates was evaluated by NMR spec-
troscopy. Due to the potential interest in this class of compounds in
PDT, photophysical and physicochemical properties of those con-
jugates were studied, including determination of singlet oxygen
production and photostability, evaluation of the electronic spectra
and aggregation studies.
(78 mg, 0.2 mmol) were dissolved in chloroform (4 mL) and stirred
under reflux for 24 h. The resulting mixture was concentrated
under vacuum and the residue was purified by silica gel column
using chloroform/methanol (99:1) as mobile phase to give 7 as a
purple solid (73 mg, 72% yield). Method B: 3 (63 mg 0.1 mmol) and
5 (78 mg, 0.2 mmol) were dissolved in chloroform (4 mL) and
placed in an ultrasonic bath (42 MHz) for 1 h at room temperature.
The resulting mixture was concentrated under reduced pressure
and the residue was chromatographed as outlined for Method A,
giving 7 with 69% yield (70 mg).
½aꢂ þ 6.25 (c 1.0, DMSO); 1H NMR (400 MHz, DMSO-d
2
5
2
. Material and methods
6
)
D
d
(ppm) 10.35 (s, 1H, NeH linker); 8.92 (d, J ¼ 4.3 Hz, 2H,
b-pyrrole),
¼ 8.5 Hz, 1H, N -H linker),
0
2.1. General procedures
8.83 (m, 6H,
8
b
-pyrrole); 8.50 (d, JHN-H1
0
.21 (d, J ¼ 5.6 Hz, 6H, o-phenyl); 8.18 (d, J ¼ 8.4 Hz; 2H, o-phenyl
The 1H NMR, C NMR and 2D experiments (HMBC- H/13,
13
1
linker); 7.96 (d, J ¼ 8.4 Hz; 2H, m-phenyl linker); 7.84 (m, 9H, m-
1
1
0
NOESY H/ H) were obtained with a Bruker AVANCE III 400
spectrometer with the indicated solvents operating at 400.15 MHz
and p-phenyl); 6.04 (t, JH1
0
-HN ¼ 8.5 Hz, 1H, H-1 ); 5.45 (t, 1H, JH2
0
-
0
0
0
¼ 9.5 Hz, 1H, H-3 ); 5.15 (t, JH2
0
0
¼ 9.5 Hz, 1H, H-2 ); 5.03 (t,
H3
-H3
0
0
0
and 100.62 MHz respectively. Chemical shifts (
d
) are expressed in
J
J
H4
0
-H5
0
¼ 9.7 Hz, 1H, H-4 ); 4.27e4.13 (m, 2H, H-5 e H-6a ); 4.07 (m,
0
parts per million (ppm) and coupling constants (J) in Hertz (Hz)
using residual solvent peaks as internal standards. Low resolution
mass spectra were obtained with a Bruker MALDI-TOF spec-
trometer with HCCA matrix. The high resolution mass spectra
6a-6b ¼ 12.0 Hz, H-6b ); 2.09e1.99 (4s, 12H, CH
3
); ꢀ2.89 (s, NH-
13
6
pyrrole). C NMR (101 MHz, DMSO-d ) d (ppm)181.9 (C]S);
170.1; 169.9; 169.6; 169.4 (4C, C]O); 141.2; 138.7; 137.5; 135.2;
134.5; 131.4; 128.1; 127.0; 121.5; 120.0; 119.6 (44C, tetraar-
ylporphyrin); 81.4; 72.9; 72.3, 70.6; 68.0; 61.8 (6C, sugar moiety);
(
HRMS) were recorded with two different spectrometers: (a) a
Bruker MicroTOF-Q II XL operating in the positive-ion mode with
20.6; 20.5; 20.4; 20.3 (4C, CH
(5.38), 516 (4.04), 551 (3.70), 590 (3.52), 646 (3.38) nm. IR (KBr):
3 3
).UVeVis (CHCl ) lmax (log ε) 419
ꢀ
1
porphyrins at 100
mg mL in methanol using sodium formate

J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
71
max 3315, 1751, 1704, 1515, 1222 cm 1. HRMS: m/z calc. for [MþH]þ,
ꢀ
the desired product 10 (Rf ¼ 0.37) with 49% yield (49 mg). Method
B: 4 (63 mg 0.1 mmol), 5 (78 mg, 0.2 mmol) and triethylamine (3 L,
.02 mmol) were dissolved in chloroform (4 mL) and placed in an
n
C
þ
H
59 51
N
6
O
9
S : 1019.3433; found: 1019.3433.
m
0
2
.2.2. Synthesis of 5-[4-((methylbenzene)thioureido)-phenyl]-
ultrasonic bath (42 MHz) for 1 h at room temperature. The resulting
mixture was dried under reduced pressure and the residue chro-
matographed as described for Method A (10, 40 mg, 40% yield).
10,15,20-triphenylporphyrin (8)
5
-(4-Aminophenyl)-10,15,20-triphenylporphyrin
3
(63 mg
2
5
1
0
.1 mmol) and benzyl isothiocyanate 6 (150 mg, 1.0 mmol) were
½aꢂ þ 5.85 (c 1.0, DMSO); H NMR (400 MHz, DMSO-d
6
)
D
dissolved in chloroform (4 mL) and stirred under reflux for 24 h.
The resulting mixture was dried under vacuum and the resulting
residue was chromatographed on a silica gel column with ethyl
d
(ppm) 9.97 (br, 1H, NeH linker); 8.92 (d, J ¼ 4.6 Hz, 2H,
b
-pyrrole),
8.82 (m, 6H,
b-pyrrole); 8.22 (d, 6H, o-phenyl); 8.02 (d, Jo-
m
¼ 8.4 Hz; 2H, o-phenyl linker); 7.84 (m, 9H, m- and p-phenyl);
acetate/hexanes (1:1) as mobile phase (40 mg, 52% yield).
7.22 (d, J ¼ 8.4 Hz; 2H, m-phenyl linker); 5.61 (d, JH1-H2 ¼ 8.7 Hz, 1H,
1
0
0
0
H NMR (400 MHz, DMSO-d
6
)
d
(ppm) 10.06 (s, 1H, NeH linker);
-pyrrole); 8.53 (t,
H-1 ); 5.34 (t,1H, JH2-H3 ¼ 9.6 Hz,1H, H-3 ); 5.01 (m, 2H, H-2 and H-
0
0
0
8
.93 (d, J ¼ 4.3 Hz, 2H,
b
-pyrrole), 8.83 (m, 6H,
b
4 ); 4.13 (m, 2H, H-5 e H-6a’); 4.05 (d, J6a-6b ¼ 11.8 Hz, H-6b ), 2.08e
0
13
J
NH-CH2 ¼ 5.3 Hz, 1H, N eH linker), 8.20 (m, 6H, o-phenyl); 8.17 (d,
1.97 (4s, 12H, CH
3
); ꢀ2.87 (s, NH-pyrrole). C NMR (101 MHz,
J ¼ 8.3 Hz; 2H, o-phenyl linker); 7.97 (d, J ¼ 8.3 Hz; 2H, m-phenyl
linker); 7.81 (m, 9H, m- and p-phenyl); 7.46 (d, Jo-m ¼ 7.3 Hz, 2H, o-
benzyl); 7.40 (t, Jm-p ¼ 7.5 Hz, 2H, m-benzyl); 7.30 (t, Jm-p ¼ 7.5 Hz,
DMSO-d (ppm) 166.9; 166.4; 166.1; 165.9 (4C, C]O); 154.4 (C]
6
) d
O, linker) 138.2; 137.9; 132.5; 131.1; 128.4; 125.0; 123.9; 117.6;
116.8; 116.6; 110.9 (44C, tetraarylporphyrin); 79.2; 69.8; 68.5; 68.3;
1
H, p-benzyl); 4.90 (d, JNH-CH2 ¼ 5.3 Hz, 2H, CH
2
); ꢀ2.87 (s, NH-
(ppm) 181.0 (C]S);
41.2; 139.4; 139.0; 136.7; 134.6; 131.4; 128.4; 128.1; 127.5; 127.0;
21.0; 120.0; 119.96; 119.90 (50C, tetraarylporphyrin and aromatic
64.5; 58.5 (6C, sugar moiety); 17.4; 17.36; 17.31; 17.2 (4C, CH
3
). UVe
13
pyrrole). C NMR (101 MHz, DMSO-d
1
1
6
)
d
Vis (CHCl max (log ε) 419 (5.37), 516 (3.98), 552 (3.67), 590 (3.45),
3
) l
ꢀ
1
646 (3.34) nm. IR (KBr):
n
max 3315,1751,1222 cm . HRMS: m/z calc.
þ
þ
for [MþH] , C59
H
50
N
5
O
11: 1004.3501; found: 1004.3457.
benzyl); 47.3 (CH
2
). UVeVis (CHCl
3
)
lmax (log ε) 420 (5.24), 516
(
n
C
3.84), 551 (3.51), 590 (3.51), 646 (3.18), 710 (2.62) nm. IR (KBr):
2.2.5. Synthesis of 5-(phenoxy-4-thiocarbonyl-
aminomethylbenzene)-10,15,20-triphenylporphyrin (11)
5-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin 4 (63 mg
ꢀ
1
þ
max 3315, 1701, 1517 cm
.
HRMS: m/z calc. for [MþH] ,
þ
H
52 39
N
6
S : 779.2951 found: 779.2952.
0
.1 mmol), benzyl isothiocyanate 6 (150 mg, 1.0 mmol) and trie-
0
2
2
.2.3. Synthesis of 5-[phenoxy-4-thiocarbonyl-(1 -deoxyamino-
thylamine (3 mL, 0.02 mmol) were dissolved in chloroform (4 mL)
0
0
0
0
,3 ,4 ,6 -tetra-O-acetyl-
b
-D-glucopyranosyl)]-10,15,20-
and stirred under reflux for 48 h. The resulting mixture was dried
under reduced pressure and the residue was chromatographed on
silica gel column with chloroform as mobile phase. The first chro-
matographic fraction eluted (Rf ¼ 0.70) gave 11 with 20% yield
(16 mg).
triphenylporphyrin (9)
-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin
.1 mmol), glucosyl isothiocyanate 5 (78 mg, 0.2 mmol) and trie-
thylamine (3 L, 0.02 mmol) were dissolved in chloroform (4 mL)
5
4 (63 mg
0
m
and stirred under reflux for 48 h. The resulting mixture was dried
under reduced pressure and the residue was chromatographed on a
column of silica gel (10 ꢃ 500 mm) with chloroform/ethyl acetate
1H NMR (400 MHz, DMSO-d
6
) d (ppm) 10.55 and 10.34 (2t, JHN-
CH2 ¼ 5.9 and 6.2 Hz, respectively, 1H, NeH linker); 8.84 (m, 8H,
b-
pyrrole); 8.21 (m, 8H, o-phenyl); 7.82 (m, 9H, m- and p-phenyl);
7.52 (d, Jo-m ¼ 8.4 Hz); 7.46 (m, 2H, o-benzyl); 7.41 (t, Jm-p ¼ 7.7 Hz,
2H, m-benzyl); 7.33 (t, Jm-p ¼ 7.7 Hz, 1H, p-benzyl); 4.87 and 4.71
(
40:1) as mobile phase. The first fraction eluted (Rf ¼ 0.44) was the
desired product (9, 23 mg, 23% yield).
2
5
1
½
aꢂ þ 4.31 (c 1.0, DMSO); H NMR (600 MHz, DMSO-d
6
)
d
(ppm)
0.82 and 10.59 (2d, JHN-H1 ¼ 9.0 and 8.6 Hz, respectively, 1H, NeH
linker), 8.83 (m, 8H,
-pyrrole); 8.24 (d, J ¼ 8.2 Hz; 2H, o-phenyl
linker), 8.21 (d, 6H, o-phenyl); 7.82 (d, 9H, m- and p-phenyl); 7.51
d, Jo-m ¼ 8.2 Hz; 2H, m-phenyl linker); 5.97 and 5.93 (2t, JHN-
(2d, JHN-CH2 ¼ 5.9 and 6.2 Hz, respectively, 2H, CH
2
); ꢀ2.89 (s, 2H,
D
13
1
NH-pyrrole). C NMR (101 MHz, DMSO-d ) d (ppm) 189.3 (C]S);
6
b
153.3; 141.2; 138.5; 137.6; 134.9; 134.2; 131.6; 128.5; 128.1; 127.6;
127.4; 127.0; 121.4; 120.1; 119.2; (50C, tetraarylporphyrin and ar-
omatic benzyl); 48.7 (CH ). UVeVis (CHCl ) lmax (log ε) 419 (5.40),
2 3
515 (4.08), 551 (3.73), 590 (3.56), 645 (3.43) nm. HRMS: m/z calc. for
[MþH] , C52
(
0
0
H1 ¼ 9.0 and 8.6 Hz, respectively, 1H, H-1 ); 5.51 (t, 1H, H-3 ); 5.16 (t,
0
0
0
þ
þ
1
1
H, H-2 ); 5.01 (t, 1H, H-4 ); 4.26 (m, 2H, H-5 and H-6a’), 4.11 (m,
38 5
H N OS : 780.2792; found: 780.2841.
0
H, H-6b ); 2.09; 2.06; 2.04; 2.00 (4s, 12H, CH
3
); ꢀ2.87 (s, NH-
(ppm) 183.1 (C]S);
70.5; 169.9; 169.6; 169.5 (4C, C]O); 153.3; 141.6; 141.5; 135.4;
34.7; 131.9; 128.6; 127.4; 121.8; 120.6; 119.5 (44C, tetraar-
ylporphyrin); 82.7; 73.2; 72.0, 71.8; 68.0; 62.0 (6C, sugar moiety);
0.9; 20.8; 20.7; 20.67 (4C, CH ). UVeVis (CHCl max (log ε) 419
13
pyrrole). C NMR (101 MHz, DMSO-d
1
1
6
)
d
2.2.6. Synthesis of 5-(phenoxy-4-carbonyl-aminomethylbenzene)-
10,15,20-triphenylporphyrin (12)
5-(4-Hydroxyphenyl)-10,15,20-triphenylporphyrin
0.1 mmol) benzyl isothiocyanate 6 (150 mg, 1.0 mmol) and trie-
thylamine (3 L, 0.02 mmol) were dissolved in chloroform (4 mL)
4 (63 mg
2
3
3
)
l
m
(
5.34), 516 (3.93), 552 (3.65), 591 (3.41), 646 (3.26) nm. HRMS: m/z
and stirred under reflux for 24 h. NaOH (4 mg, 0.1 mmol) was then
added to the reaction flask and resulting mixture was kept for
additional 24 h under stirring at room temperature. The resulting
mixture was dried under reduced pressure and the residue was
applied to a silica gel column with chloroform as mobile phase.
Chromatography gave only one fraction (Rf ¼ 0.59), which was the
þ
þ
calc. for [MþH] , C59
H
50
N
5
O10S : 1020.3273; found: 1020.3292.
0
2
2
.2.4. Synthesis of 5-[phenoxy-4-carbonyl-(1 -deoxyamino-
0
0
0
0
,3 ,4 ,6 -tetra-O-acetyl-b-D-glucopyranosyl)]-10,15,20-
triphenylporphyrin (10)
Method A: 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin 4
63 mg 0.1 mmol), glucosyl isothiocyanate 5 (78 mg, 0.2 mmol) and
desired product (12) with 32% yield (24 mg).
1
(
6
H NMR (600 MHz, DMSO-d ) d (ppm) 8.83 (m, 8H, b-pyrrole);
triethylamine (3
m
L, 0.02 mmol) were dissolved in chloroform
8.57 (t, JNH-CH2 ¼ 6.1 Hz, 1H, NeH linker); 8.20 (m, 8H, o-phenyl);
7.82 (m, 9H, m- e p-phenyl); 7.56 (d, Jo-m ¼ 8.4 Hz, 2H, m-phenyl
linker); 7.43 (m, 4H, m- and o-benzyl); 7.31 (t, Jm-p ¼ 7.0 Hz, 1H, p-
(
4 mL) and stirred under reflux for 24 h. NaOH (4 mg, 0.1 mmol) was
then added to the reaction flask and the resulting mixture was kept
for additional 24 h under stirring at room temperature. Reaction
was concentrated under vacuum and the residue was chromato-
graphed on a silica gel column with chloroform/ethyl acetate (40:1)
as mobile phase. The second chromatographic fraction eluted was
benzyl); 4.41 (d, JHN-CH2 ¼ 6.1 Hz, 2H, CH
2
); ꢀ2.91 (s, NH-pyrrole).
(ppm) 154.8 (C]O); 151.2; 141.2;
139.3; 137.8; 135.0; 134.2; 128.5; 128.1; 127.3; 127.0; 120.2; 120.1;
119.3; (50C, tetraarylporphyrin and aromatic benzyl); 44.3 (CH ).
13
6
C NMR (101 MHz, DMSO-d ) d
2

72
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
UVeVis (CHCl
3
)
l
max (log ε) 419 (5.40), 515 (4.06), 550 (3.68), 590
Table 1
ꢀ1
Optimizationof the reaction conditions for the synthesisof thioureaglycoporphyrin 7.
(
3.52), 645 (3.35) nm. IR (KBr):
n
max 3317, 1730, 1207 cm . HRMS:
þ
þ
m/z calc. for [MþH] , C52
.3. Singlet oxygen measurements
Singlet oxygen production was estimated through the indirect
H
38
N
5
O
2
: 764.3020 found: 764.3033.
2
method described by Roitman and co-workers [36] with modifi-
cations. Freshly prepared solutions of 1,3-diphenylisobenzofuran
ꢀ
1
ꢀ1
(
DPBF) (50
m
mol L ) and the tested porphyrin (0.5
O (9:1) were irradiated
with 9.0 mW/cm with a Lumacare light source using a yellow filter
550e800 nm) at room temperature, under stirring for different
mmol L , pre-
pared from the stock solutions) in DMF:H
2
2
(
periods of time (from 0 to 30 min; see Fig. 4). The same procedure
was performed for the standard photosensitizer, methylene blue
1
(
MB). The reaction of DPBF with O
2
was monitored by the reduc-
tion of intensity of the absorption band at 415 nm over time. The
absorbance values were measured twice in independent experi-
ments and the results were expressed in percentage with the cor-
responding standard deviation. The singlet oxygen quantum yields
were obtained from Equation (1) using just the first eight minutes,
which corresponded to range of time that presented a linear rela-
tionship with the natural logarithm (ln) of the DPBF absorbance.
ꢁ
Yield (%) 7b
Entry
5 (equiv)
Time (h)
T ( C)
Et
3
N (equiv)
a
1
2
3
4
5
6
7
2
10
2
2
2
4
4
r.t.
r.t.
r.t.
Reflux
Reflux
Reflux
Reflux
e
e
e
e
e
10
15
54
72
70
a
a
24
24
48
48
1
a
a
a
c
2
2
0.2
e
60
Std
R$I
d
Std
D
abs
69
FD
¼
F
(1)
RStd$Iabs
a
Reactions performed with 3 (0.1 mmol), in 2 mL of CHCl3.
Yields estimated from flash chromatography.
Traces of glycoporphyrin bearing an urea group were detected.
b
c
In Equation (1),
F
D
represents the oxygen singlet yield of the
Std
sample, with
F
being the standard quantum yield (for MB, 0.52).
d
3
Reaction performed with 3 (0.1 mmol), in 2 mL of CHCl , ultrasound (42 MHz).
D
R and RStd are the rate constants of DPBF consumption in the
presence of porphyrins and MB, respectively. Since it is a first order
reaction, these parameters were obtained from a plot of the ln of
Std
abs
DPBF consumption. Iabs and I are the rates of light absorption for
the sample and the standard, respectively [29].
2.4. Photostability studies
The photostability evaluation was performed by Gomes and co-
ꢀ
1
workers method [37]. Aqueous solutions of porphyrin (10 mmol L ,
prepared from the stock solutions) were irradiated with 100 mW/
2
cm using Lumacare light source at room temperature under stir-
ring for different periods of time (from 0 to 30 min, see Fig. 6). To
avoid porphyrin aggregation, 7 and 9 were dissolved in an SDS
solution (0.1 mg %). The results were monitored in terms of the
Scheme 1. Synthesis of mono-substituted amino and hydroxyl meso-tetra(aryl)
porphyrins 3 and 4.
Scheme 2. Synthesis of thiourea benzyl-porphyrin 8.

J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
73
reduction of Soret band absorption intensity over time. The
extinction of Soret band was measured twice with independent
experiments and the results were expressed in percentage with the
correspondent standard deviation.
our delight, the reaction time was reduced to 1 h with no sub-
stantial decreasing in reaction yield.
As one of our main objectives was to understand the effect of the
linker on the physicochemical and photophysical properties of the
resulting porphyrin conjugates, we attempted to discriminate the
influence of the carbohydrate moiety on such properties. For this
purpose, we also synthesized a non-glycosyl thiourea porphyrin
conjugate. Specifically, we prepared benzyl isothiocyanate 6 from
benzyl bromide [47], which was in turn submitted to the optimized
protocol with amino-tetra(aryl)porphyrin 3 (conditions identical as
indicated in entry 4 of Table 1, except for the addition of 10 equiv. of
benzyl isothiocyanate 6) for the synthesis of hitherto unreported
porphyrin 8 (Scheme 2). Attempts to reduce the reaction time using
ultrasound were unsuccessful, and just traces of target porphyrin 8
were detected.
2.5. Aggregation studies
Aqueous solutions of the porphyrins in different concentrations
were prepared from the stock solutions, as indicated in Figs. 7 and
. Solutions were then measured by means of UVeVis absorption
8
and fluorescence emission. As previously described [38], porphyrin
self-assembling behavior was evaluated through the observation
maximum absorbance or emission shifting in function of the
porphyrin concentration.
3
. Results and discussion
Table 2
Reaction conditions for the synthesis of thiocarbamate 9 and carbamate 10 glyco-
3.1. Chemistry
porphyrins.
In the present work, the synthesis of porphyrin conjugates was
planned based on the use of isothiocyanate derivatives and asym-
metric meso-tetra(aryl)porphyrins containing one amino or hy-
droxyl group. To synthesize the mono-substituted porphyrins, we
decided to make a brief study on different synthetic routes aiming
to better overall yields. This study was performed by reproducing
some literature porphyrin synthesis [39e42] and by slightly
modifying established methodologies [31,40,43] (for details see
Supplementary Information). The best overall yield for amino-
tetra(aryl)porphyrin 3 synthesis was achieved via the correspond-
ing nitro-tetra(aryl)porphyrin 1 route. For this purpose, we first
performed a cyclocondensation reaction of pyrrole, benzaldehyde
and p-nitrobenzaldehyde followed by porphyrinogen oxidation
2
with SeO , an oxidizing agent that has been recently introduced by
us for porphyrin synthesis [40]. Nitro compound 1 was then
0
reduced to amine 3 using Sn as reducing agent under acid con-
ditions (Scheme 1). The route selected for the synthesis of hydroxy-
tetra(aryl)porphyrin 4 involved aqueous acid hydrolysis of acetoxy-
tetra(aryl)porphyrin 2. In contrast to porphyrin 1 synthesis, com-
pound 2 best yield was achieved in conditions having DDQ (2,3-
dichloro-5,6-dicyano-1,4-benzoquinone), instead of SeO
oxidizing agent (Scheme 1).
2
,
as
Diverse approaches have been reported for the preparation of
glycosyl isothiocyanate building blocks [44e46]. Here, compound 5
was synthesized from acetobromoglucose through bromide
nucleophilic displacement with KSCN [34]. Having both glycosyl
isothiocyanate and porphyrin starting materials, we initiated an
optimization study with 5 and porphyrin 3 to prepare the thiourea
glycoporphyrin 7 (Table 1). Product (7) structure was confirmed
unambiguously by the expected spectral features, including the
13
deshielded position of C]S signal in the C NMR spectra at
81.9 ppm. Reaction conditions using room temperature for four
1
hours (Table 1, entry 1) gave 7 in low yield (10%). Even utilizing
increased amounts of the isothyocyanate 5, no important yield
enhancement was achieved (entry 2). We then decided to use
longer reactions times, as well as a higher temperature (entries 3e
ꢁ
Yield (%) 9/10b
Entry
5 (equiv)
Time (h)
T ( C)
Et
3
N (equiv)
5
). The resulting conditions improved the reaction outcome and
a
glycoporphyrin 7 was obtained with 72% yield by using 24 h re-
action under reflux (entry 4). A condition attempting the using of
Et N as catalyst showed an acceptable yield (entry 6); however,
3
traces of a porphyrin glycoconjugate bearing a urea group was
identified by mass spectroscopy analysis (see Supplementary
Information). With the objective of reducing the reaction time,
we submitted a reaction mixture identical as that outlined for entry
1
2
3
4
2
2
2
2
48
48
1
Reflux
Reflux
r.t.
e
19/e
23/33
8/40
a
0.2
0.2
0.2
c
d
48
Reflux
e/49
a
Reactions performed with 4 (0.1 mmol), in 2 mL of CHCl3.
Yields estimated from flash chromatography.
Reaction performed with 4 (0.1 mmol), in 2 mL of CHCl
Reactions performed with 4 (0.1 mmol), in 2 mL of CHCl3. Then NaOH (1 equiv)
b
c
3
, ultrasound (42 MHz).
d
4
to ultrasound irradiation under room temperature (entry 7). To
was added for 24 h under room temperature.

74
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
Scheme 3. Synthesis of thiocarbamate (11) and carbamate (12) benzyl-porphyrins.
Addition reactions of hydroxyl compounds to O-protected
Evaluation of the 1H NMR spectra of compounds 7e12 was
conducted considering the fact that thiuorea, thiocarbamate and
carbamate units are not rigid linkers, but they can experience a
reduction of the rotation of the CeN pseudo-amide bond [55e57].
For this reason, these compounds could generate NMR spectra
consistent with the presence of distinct populations of conformers.
According to the literature [26] due to the greater bulk and polar-
izability of sulfur in comparison to oxygen, the rotational energetic
barrier is typically higher for N-thiocarbonyls than for the carbonyl
analogs. When comparing the N-thiocarbonyl counterparts, the
energy related to the rotation of the N-group out of the plane is
glycosyl isothiocyanates have been often reported for the synthesis
of thiocarbamate glycoconjugates [48,49]. Due to the lower nucle-
ophilicity of hydroxyl in relation to amino groups, some protocols
have been optimized with bases as catalysts to perform such re-
actions [50]. However, carbamate derivatives have been produced as
byproducts under those conditions [51]. Notwithstanding this
observation, the sulfureoxygen exchange related to thiocarbamates
can be synthetically useful. This is based on the fact that the obvious
starting materials for carbamate synthesis (isocyanates) rapidly
subside to hydrolysis upon exposure to moisture. Because isothio-
cyanate compounds are easier to handle [23], their employment as
starting materials for the synthesis of carbamates have been recently
explored [52]. In the present work, we exercised reaction versatility
by synthesizing both porphyrin thiocarbamate 9 and porphyrin
carbamate 10 from hydroxy-tetra(aryl)porphyrin 4 and glycoside 5
1
usually higher for thiocarbamate than for thiourea. Here, H NMR
spectra of each of the thiourea compounds 7 and 8 gave only one
signal pattern, indicating the absence of detectable additional
conformers. This behavior could be either related to (a) a fast
interconversion among the possible conformers under NMR con-
ditions herein utilized or (b) the presence of a highly predominant
(
Table 2). We first applied the protocol that proved to be the most
efficient based on the synthesis of the thiourea compounds 7 and 8
to give thiocarbamate 9 with 19% yield (Table 2, entry 1). The base
catalyzed reaction shown in entry 2 (Table 2) also produced 9,
although in lower yield in relation to carbamate porphyrin 10. The
more stable conformation. It is also important to mention that the
1
H
NMR temperature of coalescence for thiourea conformers
ꢁ
ranges from 0 to 40 C [26], with our NMR experiments being
conducted at 30 C. Despite of those facts, per-O-acylated glyco-
ꢁ
13
linker structure could be discriminated for both products by
C
sylthioureas tend to adopt Z configuration at the sugar NHeC(]S)
bond. Thiourea glycoporphyrin 7 gave a coupling constant of 8.5 Hz,
which is in agreement with the expected anti relationship for NeH
and sugar H-1. This is also in accordance with the relatively shiel-
ded anomeric hydrogen (6.04 ppm) presented by 7. For compound
8, the coupling constant related to NeH and benzylic hydrogens
was 5.3 Hz, this value being more consistent with a gauche relative
disposition involving the three mentioned hydrogens. Compounds
NMR spectroscopy. In agreement with literature data [28,53,54], the
C]S carbon of 9 was assigned at 183.1 ppm and the C]O carbon of
10 at 154.4 ppm. Reaction using ultrasound irradiation under room
temperature (entry 3) gave mainly the carbamate porphyrin 10 at a
reduced reaction time (1 h). At this stage, we planned a protocol to
exclusively obtain the carbamate derivative 10, which was devel-
oped byadding NaOH and keeping the resulting reaction mixture for
1
1
2
4 h at room temperature (entry 4).
7 and 8 were then submitted to NOESY He H NMR experiments.
These experiments gave no interactions involving m-phenyl hy-
drogens of the tetra(aryl)pophyrin moiety and the HeN closer to
the sugar ring, which excludes the possibility of an E conformation.
A NOESY cross peak between the two HeN of the thiourea bridge
could also be observed. These findings, together with the above-
cited literature data, indicated the thiourea bridge as having a Z,Z
conformation for both 7 and 8.
With the above information to hand, the synthesis of benzyl
thiocarbamate and carbamate porphyrins 11 and 12 were per-
formed as shown in the Scheme 3. Attempts to reduce reaction time
using ultrasound were unsuccessful, and just traces of target por-
phyrins 11 and 12 were found. Considering the fact that the
ultrasound-assisted reactions only gave good results for the syn-
thesis of the glycoporphyrins, we concluded that the type of the
isothycianate utilized as starting material seemed to have a pivotal
importance for the success of the ultrasound protocol.
Differently, for each of the thiocarbamate derivates (9 and 11),
two different conformer populations could be observed. Compound

J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
75
Fig. 1. Selected H1 NMR signals of HeN and glycosyl-H-1 or benzyl-CH
of compounds 9 (a) and 11 (b).
2
9
3
had two doublets at 10.82 and 10.59 ppm (integration ratio of
.9:1, respectively), which were assigned to HeN of the thio-
1H NMR spectrum of thiocarbamate 11 presented two triplets
from HeN (10.55 and 10.34 ppm) and two doublets from benzylic
carbamate bridge of the two distinct conformers. These signals had
matching coupling constants of 9.0 and 8.6 Hz related to a multiplet
2
CH (4.86 and 4.71 ppm), also having matching coupling constants
of 5.9 and 6.2 Hz, respectively. The ratio between the conformers
was higher for thiocarbamate benzyl-porphyrin (5.6:1) than for
glycoporphyrin analog (Fig. 1b). The evaluation of the coupling
constants allowed us to assign a Z conformation between C]S and
(
which corresponded to two partially overlapped triplets) attrib-
uted to the anomeric hydrogen (H-1) of the glycosyl moiety at
.97e5.90 ppm (see Fig. 1a). Selective irradiation of HeN of 9 major
5
conformer, at 10.82 ppm, gave NOE peaks at 5.16 and 5.97 ppm (H-2
and H-1 of the sugar moiety, respectively). Together with the
characteristic coupling constants, this data suggested that the NHe
C(]S) bond had Z configuration. The appearance of another NOE
signal at 7.51 ppm (m-phenyl hydrogens of the tetra(aryl)pophyrin
moiety) defined the thiocarbamate bridge of 9 major conformer as
Z,E. In turn, 9 minor conformer had only one HeN NOE signal
attributed to H-2 of the sugar moiety. The lack of further NOE peaks
could be a result of the low abundance of the conformer, which
compromised NMR signal gain. Considering chemical shifts and
coupling constant values, a minor conformer of 9 should present a
Z,Z configuration.
2
CH for both conformers. Considering the absence of NOE at
7.52 ppm (m-phenyl hydrogens of the tetra(aryl)pophyrin moiety)
we could propose a Z,Z conformation for the major population of
compound 11. Assuming that compound 11 major and minor con-
formers should present distinct conformations from each other, we
could speculate a Z,E conformation for the minor conformer. The
lack of an NOE signal involving the m-phenyl hydrogens of the
tetra(aryl)porphyrin moiety could also be explained by low NMR
signal gain, as noticed for compound 9 minor conformer.
It was noteworthy that porphyrin conjugates 9 and 11 were
unstable in terms of their thiocarbamate linkages. For example,
even storing a DMSO solution of 9 in freezer for only three days, it

76
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
1
Fig. 2. Most likely conformations of compounds 7, 8, 9 (major conformer), 11 (major conformer) and 12. Key H NMR chemical shifts (
d
) and coupling constant values (Hz) are
) represent observed 1D 1H NMR NOE peaks; two-headed arrows (
) represent observed 2D He H NMR NOESY cross peaks and dashed lines
1
1
shown. One-headed arrows (
) represent non-observed NOE peaks or NOESY cross peaks.
(
could be noted the appearing of small amounts of porphyrins 4 and
0. The hydrolysis of the thiocarbamate derivates was somewhat
solvent provided proper solubilization for all porphyrin conjugates.
We anticipated that specifically for the carbamate bridge of the
glycoporphyrin 10, a substantial HeN hydrogen exchange with the
water present in the deuterated solvent could have occurred. This
supposition was supported by a presat H NMR experiment (water
signal suppression using presaturation), which concomitantly
1
expected because previously studies have already reported this
type of behavior under both acid or basic conditions [25]. For this
reason, attempts of prolonged or temperature-based NMR experi-
1
1
ments, such as NOESY 2D and dynamic H NMR, gave disappoint-
ingly results due to decomposition during experiment course. In
this way, the following physicochemical and photophysical assays
were performed using freshly purified 9 and 11 samples.
The carbamate benzyl-porphyrin 12 HeN signal appeared as a
triplet (8.57 ppm, J ¼ 6.1 Hz) with matching coupling constant with
a doublet that corresponded to the benzylic hydrogens (4.41 ppm).
The close J value to those observed for the other benzyl-porphyrins
eliminated the HeN signal. The expected position and configura-
1
13
tion of the OeC(]O)eNH group was confirmed by HMBC He
C
NMR that displayed correlations at 5.61/154.4 ppm (H-1/C]O) and
9.97/69.8 ppm (HeN/C-2). Because of the absence of coupling
constants, as well as the lack of any NOESY cross peak involving
carbamate bridge of 10, we could not propose any possible
conformation for this compound. Fig. 2 present the most likely
conformations expected for compounds 7e9, 11 and 12 based on
(8 and 11) indicated that HNebenzyl as having Z conformation. The
1
lack of an NOESY cross peak involving HeN and m-phenyl hydro-
gens of the tetra(aryl)pophyrin moiety defined the 12 carbamate
bridge with Z,Z conformation.
literature data, H NMR chemical shifts, coupling constant values
and NOE or NOESY experiments (see spectra in Supplementary
Information).
13
Although carbamate derivative 10 presented mass and C NMR
spectra compatible to the expected structure, the 1H NMR signals
related to the carbamate bridge showed an unexpected multiplic-
ity. Specifically, HeN appeared as a broad signal, while anomeric
hydrogen of the glycosyl moiety gave a doublet (it was expected a
doublet and a triplet, respectively). All the NMR experiments
above-discussed were conducted in deuterated DMSO because this
3.2. Evaluation of the photophysical and physicochemical
properties of porphyrins 7e12
The evaluation of photophysical and physicochemical properties
of a PS is current utilized to identify promising molecules for PDT
mol L 1 in CHCl
ꢀ
Fig. 3. UVeVis spectra of Q-bands of porphyrins 7e12 at 12
m
3
.
Inset:Soret band.
Fig. 4. Singlet oxygen production of 7e12.

J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
77
Fig. 5. DPBF consumption in the presence of MB in DMF:H
2
O 9:1 at 9 mW/cm2 (inset:
ln (DPBF absorbance) vs irradiation time).
[29,58]. In order to identify whether there was any effect related to
the thiourea, thiocarbamate and carbamate bridges on porphyrin
electronic spectra, the UVeVis spectral profile of porphyrins 7e12
and synthetic precursors 3 and 4 were recorded. Fig. 3 shows the
UVeVis data for all porphyrins studied. Except for porphyrin 8, the
UVeVis spectra were very similar for all compounds. For porphyrin
8
, it was found an additional Q-band at a higher wavelength
(
710 nm). This could be explained by the fact that NeC(]S)eN
group shows a preference for polar resonance structures, with the
sulfur atom being able to bear a negative charge by making a
transitory single bond to the central carbon [26]. In this case,
benzylic hydrogens could become more acidic, thus creating the
opportunity to extend the aryl-porphyrin group resonance to the
benzene ring of the deprotonated benzyl group.
Even though the new band in compound 8 UVeVis spectrum
presented low intensity, we considered this finding important.
Meso-tetra(aryl)porphyrins have multiple potential sites for placing
amino groups, allowing the introduction of additional thiourea
bridges. This strategy could be used in the future in order to amplify
the absorptivity in the spectral range of 700e850 nm. In such
wavelength region, the light shows deeper tissue penetration,
which makes possible an increased singlet oxygen production in
those tissues and, consequently, increasing PDT efficacy [3,26].
Fig. 6. Photostability study. Porphyrin concentration 10
m
M on aqueous media; (a)
Soret band extinction in the absence of light on aqueous media. (b) Soret band
2
extinction under irradiation at 100 mW/cm ; Obs: for graphic (b) 7 and 8 were dis-
solved in a 0.1 mg% SDS aqueous solution.
Compounds 10 and 12, both carbamate porphyrin derivates (green
1
2
1
2
lines e Fig. 4), were the best O producers. Thiourea compounds 7
and 8 gave higher amounts of O than compound 3 (their synthetic
precursor). In turn, thiocarbamate derivates 9 and 11 had close
results in comparison to their synthetic precursor (porphyrin 4).
One of the characteristics that are expected from an efficient PS
1
is the ability of generating high amounts of singlet oxygen ( O
2
). We
1
2
estimated O production for porphyrins 7e12 and their synthetic
precursors, compounds 3 and 4, by a previously stated methodol-
ogy. In this indirect method we measured the decreasing of the
absorption of DPBF (1,3-diphenylisobenzofuran) at 415 nm in the
presence of the studied porphyrins. We reported the UVeVis ab-
sorption decreasing in terms of percentage from the initial values;
1
thus lower percentages reflect a higher O
2
production.
Results outlined in Fig. 4 indicated that there was a perceptible
1
correlation between the linker type and the
2
O production.
Table 3
Singlet oxygen quantum yield (
D
F ).
Compound
R
F
D
Methylene blue
Porphyrin 3
Porphyrin 4
Porphyrin 7
Porphyrin 8
Porphyrin 9
Porphyrin 10
Porphyrin 11
0.0333
0.0093
0.0242
0.0234
0.0132
0.0175
0.0295
0.0156
0.0297
e
0.52
0.15 ꢄ 0.051
0.38 ꢄ 0.062
0.37 ꢄ 0.046
0.21 ꢄ 0.053
0.27 ꢄ 0.056
0.46 ꢄ 0.072
0.24 ꢄ 0.067
0.46 ꢄ 0.065
0.32
Porphyrin 12
Fig. 7. UVeVis spectra with different porphyrins concentration. (a)7 (1) 5 ꢃ 10 7; (2)
ꢀ
Photofrina
ꢀ
6
ꢀ6
ꢀ6
ꢀ5
ꢀ5
ꢀ1
ꢀ6
5
ꢃ 10 ; (3) 2 ꢃ 10 ; (4) 10 ; (5) 1.5 ꢃ 10 ; (6) 10 mol L ; (b) 8 (1)1 ꢃ 10 ; (2)
ꢀ6 ꢀ6 ꢀ5 ꢀ5 ꢀ5 ꢀ1
a
Data obtained from Ref. [28].
5 ꢃ 10 ; (3) 2 ꢃ 10 ; (4) 2.5 ꢃ 10 ; (5) 1.5 ꢃ 10 ; (6) 10 mol L
.

78
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
evaluated the behavior of the porphyrins in the absence of light. As
shown in Fig. 6a, in a preliminary study we could observe that 7 and
8
had an intense decreasing of the Soret band absorption in the first
3
min. This behavior was compatible with events of molecular
aggregation. To avoid the impact of aggregation during photo-
stability studies, we first developed a surfactant system that did not
cause a significant interference on Soret band absorption. This
consisted of a diluted solution of SDS 0.1 mg/100 mL. In this way,
compounds 7 and 9 were submitted to the photostability test being
dissolved in the SDS solution instead of water. As observed for the
1
2
O production study, we found an important correlation between
the linkage and the photostability results. Thiocarbamate porphy-
rins derivates 9 and 11 were the less photostable compounds,
presenting a Soret band extinction profile very similar to their
synthetic precursor (compound 4). Thiourea derivatives 7 and 8
were more photostable than their synthetic precursor (porphyrin
3), with the carbamates 10 and 12 being the most photostable
compounds in the study (Fig. 6b).
Both 7 and 8 thiourea porphyrin conjugates aggregated in
aqueous media. We then wondered if their self-assembling prop-
erties were equal, as they were closely related compounds. So, we
evaluated them by UVeVis (Fig. 7) and fluorescence spectroscopy
(Fig. 8a) through a previously stated method based on the exciton
theory established by Kasha and coworkers [38,59,60]. According to
this theory, the shifts in UVeVis spectra and the changes in fluo-
rescence emission intensity can indicate the geometry achieved
after self-assembling events. Surprisingly, the aggregation proper-
ties were not the same for the thiourea conjugates. Whilst
increasing the concentration of porphyrin 7 resulted in a blue-shift
of the Soret band, porphyrin 8 showed a red-shifting behavior. In
agreement with the UVeVis experiment, concentration increase
produced a decrease in the fluorescence intensity for 7 and a
fluorescence enhancement for 8. This is consistent with the for-
mation of H-aggregates for porphyrin 7 and J-type aggregates for
porphyrin 8, respectively. The appearance of a shoulder band and a
relatively broad profile at half-height indicated that only part of the
porphyrins in the solution became H- or J-aggregates and, thereby,
porphyrin monomers are still present, even under high porphyrin
concentration. With these results we were able to propose a ge-
ometry arrangement for the aggregates of glycoporphyrin 7 and
benzyl-porphyrin 8, as shown on Fig. 8b.
Fig. 8. (a) Changes in fluorescence emission spectra. Plot of relative intensity area from
60 to 750 nm over concentration of 7 and 8 in aqueous solution. Circles and triangles
represent the experimental data points. (b) The proposed H- and J-aggregation
structure of the porphyrins linker;
6
7
and 8. Squares ¼ porphyrin ring; lines ¼
circles ¼ benzyl or glycosyl moiety.
4. Conclusions
1
Porphyrin O
2
quantum yields (
F
D
), were determined with MB
Here, we described the synthesis of glyco- and benzyl-
as standard photosensitizer, as previously stated [29]. By plotting ln
of DPBF absorbance during irradiation time in the presence of MB,
we were able to extract the angular coefficient, which was named
tetra(aryl)porphyrin conjugates (7e12) bearing thiourea, thio-
carbamate or carbamate linkers from monosubstituted amino- (3)
or hydroxy- (4) meso-tetra(aryl)porphyrins and per-O-acetyl-b-D-
Std
as the standard rate constant of DPBF consumption (R ) (Fig. 5).
glucopyranosyl 5 or benzyl isothiocyanates 6. Glycoporphyrins
could be obtained in better yields than their respective non-
glycosyl analogs. Thiourea conjugates were obtained in higher
yields in comparison to the thiocarbamate and carbamate com-
pounds. An ultrasound-assisted method was successfully executed
for the preparation of those glycoporphyrins having thiourea and
carbamate bridges with a drastic reduction of the reaction time.
Benzyl-porphyrins could not be prepared by the ultrasound pro-
tocol. Attempts to define bridge conformation suggested that
thiourea and carbamate linkers tend to appear as Z,Z conformers.
Thiocarbamate derivatives presented distinct populations of
detectable rotamers, which were detected by using NMR spec-
troscopy. Major conformer of the glycoporphyrin thiocarbamate
was assigned as Z,E. We also observed a high correlation between
the type of linker and photophysical properties that allowed us to
identify the carbamate porphyrin derivates as the most promising
photosensitizers among the studied molecules. Carbamate
The same procedure was also performed for each of the studied
porphyrins to give their individual rate constant of DPBF con-
sumption (R). From Equation (1), we then calculated the singlet
oxygen quantum yields, as shown in Table 3. These results indicated
1
that the carbamate derivates 10 and 12 gave the highest
duction (
2
O pro-
F
D
¼ 0.46) among the tested porphyrins, which confirmed
the experiments outlined in Fig. 4. The comparison of the data
Ò
found in the present study and the value of
D
F for Photofrin
(
F
D
¼ 0.32) [28] make compounds 10 and 12 good candidates as
photosensitizer for PDT treatments.
The photodynamic action of porphyrins also depends on the
integrity of the macrocyclic ring during light irradiation. The pho-
tostability study of porphyrins 7e12 and compounds 3 and 4 was
performed by measuring the Soret band extinction in aqueous
media. Because many factors could influence the decrease of the
Soret band, such as aggregation and chemical degradation, we first

J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
79
derivatives showed a high singlet oxygen production and good
photostability, with no self-assembling behavior in aqueous media.
Based on these results, the synthesis of new porphyrin conjugates
that combine the best structural features herein determined can be
performed. The investigation of the photodynamic biological ac-
tivity of these compounds is currently under investigation in our
laboratories.
photocytotoxicity for application in photodynamic therapy. Tetrahedron
011;67:4924e32.
19] Locos OB, Heindl CC, Corral A, Senge MO, Scanlan EM. Efficient synthesis of
glycoporphyrins by microwave-mediated “Click” reactions. Eur J Org Chem
2010;2010:1026e8.
20] Laville I, Pigaglio S, Blais JC, Loock B, Maillard P, Grierson D, et al. A study of
the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic
therapy, in human colon tumoral cell: a liquid chromatography and MALDI-
TOF mass spectrometry analysis. Bioorg Med Chem 2004;12:3673e82.
21] Lafont D, Zorlu Y, Savoie H, Albrieux F, Ahsen V, Boyle RW, et al. Mono-
glycoconjugated phthalocyanines: effect of sugar and linkage on photody-
namic activity. Photodiag Photodyn 2013;10:252e9.
22] Hermanson GT. Bioconjugate techiniques. 2nd ed. London: Elsevier Inc.; 2008.
23] Clarke OJ, Boyle RW. Isothiocyanatoporphyrins, useful intermediates for the
conjugation of porphyrins with biomolecules and solid supports. Chem
Commun 1999;1999:2231e2.
24] Rusin O, Hub M, Král V. Novel water-soluble porphyrin-based receptors for
saccharide recognition. Mater Sci Eng C 2001;18:135e40.
25] Humeres E, de Souza EP, Debacher NA. The mechanisms of hydrolysis of N-
alkyl O-arylthioncarbamate esters. J Phys Org Chem 2010;23:915e24.
[26] Garcia Fernández JM, Ortiz Mellet C. Chemistry and developments of N-thi-
ocarbonyl carbohydrate derivatives: sugar isothiocyanates, thioamides, thio-
ureas, thiocarbamates, and their conjugates. Adv Carbohydr Chem Biochem
2
[
[
[
Acknowledgments
[
[
This work was supported by grants from Fundação Araucária
(
15152), CAPES and PRONEX-Carboidratos (14669). J.C.C.D. thanks
[
[
the scholarship from CNPq (National Research Council of Brazil).
M.E.D and M.D.N are research members of CNPq. A. G. G. is a
research member of Fundação Araucária. The authors are thankful
to Professor Luciano Huergo and Dr. Lauro M. Souza for high res-
olution mass spectroscopy, to Professor Guilherme Sassaki from
UFPR NRM Center for NOE and NOESY NMR experiments and to
UFPR Vibrational Spectroscopy Laboratory for FTIR experiments.
2000;55:35e135.
[27] Guo L, Yan B. Near-infrared luminescent hybrid materials using modified
functional lanthanide (Nd3þ, Yb3þ) porphyrins complexes chemical bonded
with silica. Inorg Chem Commun 2011;14:1833e7.
Appendix A. Supplementary data
[28] Silva P, Fonseca SM, Arranja CT, Burrows HD, Urbano AM, Sobral AJFN. A new
nonconjugated naphthalene derivative of meso-tetra-(3-hydroxy)-phenyl-
porphyrin as a potential sensitizer for photodynamic therapy. Photochem
Photobiol 2010;86:1147e53.
[29] de Assis FF, de Souza JM, Assis BHK, Brocksom TJ, de Oliveira KT. Synthesis and
photophysical studies of a chlorin sterically designed to prevent self-aggre-
gation. Dyes Pigment 2013;98:153e9.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.03.029.
References
[30] Armarego WLF, Perrin DD. Purification of laboratory chemicals. 4th ed. Ox-
ford: Butterworth-Heinemann; 2000.
[
[
[
1] Brown SB, Brown EA, Walker I. The present and future role of photodynamic
[31] Kruper WJ, Chamberlim TA, Kachanny M. Regiospecific aryl nitration of meso-
substituted tetraarylporphyrins: a simple route to bifunctional porphyrins.
J Org Chem 1989;54:2753e6.
[32] Kumar D, Shekar KPC, Mishra B, Kumar A, Akamatsu K, Kusaka E, et al. A facile
synthesis and cytotoxicity of novel porphyrin-cryptolepine conjugates. Chem
Biol Interface 2012;2:114e21.
[33] Tomé JPC, Neves MGPMS, Tomé AC, Cavaleiro JAS, Mendonça AF, PegadoIN, et al.
Synthesis of glycoporphyrin derivatives and their antiviral activity against
herpes simplex virus types 1 and 2. Bioorg Med Chem 2005;13:3878e88.
[34] Camarasa JM, Fernadez-Reza P, Garcia-Lopez TM, Heras, frederico G, Mendez-
Castrillon PP, Felix AS. A new procedure for the synthesis of glycosyl iso-
thiocyanates. Synthesis 1984;1984:509e10.
[35] Kitamura T, Kobayashi S, Taniguchi H. Photolysis of vinyl halides. Reaction of
photogenerated vinyl cations with cyanate and thiocyanate ions. J Org Chem
1990;55:1801e5.
[36] Roitman L, Ehrenperb B, Kabayashi N. Spectral properties and absolute
determination of singlet oxygen production by naphthaloporphyrins.
J Photochem Photobiol A Chem 1994;77:23e8.
[37] Gomes MC, Barreira SMW, Faustino MAF, Fernandes R, Neves MG, Tomé AC,
et al. Photodynamic inactivation of Penicillium chrysogenum conidia by
cationic porphyrins. Photochem Photobiol Sci 2011;10:1735.
[38] Guo L. Side-chain-controlled H- and J-aggregation of amphiphilic porphyrins
in CTAB micelles. J Colloid Interface Sci 2008;322:281e6.
[39] Lee C-H, Lindsey JS. One-flask synthesis of meso-substituted dipyrromethanes
and their application in the synthesis of trans-substituted porphyrin building
blocks. Tetrahedron 1994;50:11427e40.
therapy in cancer treatment. Lancet Oncol 2004;5:497e508.
2] Bonnett R. Photodynamic therapy in historical perspective. Rev Contemp
Pharm 1999;10:1e17.
3] O’Connor AE, Gallagher WM, Byrne AT. Porphyrin and nonporphyrin photo-
sensitizers in oncology: preclinical and clinical advances in photodynamic
therapy. Photochem Photobiol 2009;85:1053e74.
[
4] Sanabria LM, Rodríguez ME, Cogno IS, Belén N, Vittar R, Pansa MF, et al. Direct
and indirect photodynamic therapy effects on the cellular and molecular
components of the tumor microenvironment. Biochim Biophys Acta
2013;1835:36e45.
[
[
[
[
[
5] Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic ther-
apy: part two e cellular signaling, cell metabolism and modes of cell death.
Photodiag Photodyn 2005;2:1e23.
6] Yano S, Hirohara S, Obata M, Hagiya Y, Ogura S, Ikeda A, et al. Current states
and future views in photodynamic therapy. J Photochem Photobiol C Photo-
chem Rev 2011;12:46e67.
7] Kejík Z, B ꢁr íza T, Králová J, Pou ꢁc ková P, Král A, Martásek P, et al. Coordination
conjugates of therapeutic proteins with drug carriers: a new approach for
versatile advanced drug delivery. Bioorg Med Chem Lett 2011;21:5514e20.
8] Umezawa N, Matsumoto N, Iwama S, Kato N, Higuchi T. Facile synthesis of
peptide-porphyrin conjugates: towards artificial catalase. Bioorg Med Chem
010;18:6340e50.
9] James DA, Swamy N, Paz N, Hanson RN, Ray R. Synthesis and estrogen re-
ceptor binding affinity of porphyrin-estradiol conjugate for targeted
photodynamic therapy of cancer. Bioorg Med Chem Lett 1999;9:2379e84.
2
a
[
10] Ethirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in
tumor imaging and photodynamic therapy. Chem Soc Rev 2011;40:340e62.
11] Zheng X, Pandey RK. Porphyrin-carbohydrate conjugates: impact of carbo-
hydrate moieties in photodynamic therapy (PDT). Anticancer Agents Med
Chem 2008;8:241e68.
[40] Ló SMS, Ducatti DRB, Duarte MER, Barreira SMW, Noseda MD, Gonçalves AG.
Synthesis of meso-tetraarylporphyrins using SeO
2011;52:1441e3.
2
as oxidant. Tetrahedron Lett
[
[41] Osati S, Safari N, Jamaat PR. Synthesis and characterization of three covalently
linked porphyrin-phthalocyanine pentamers with nucleophilic substitution.
Inorg Chim Acta 2010;363:2180e4.
[
[
[
[
[
12] Jain K, Kesharwani P, Gupta U, Jain NK. A review of glycosylated carriers for
drug delivery. Biomaterials 2012;33:4166e86.
13] Rambaruth NDS, Dwek MV. Cell surface glycan-lectin interactions in tumor
metastasis. Acta Histochem 2011;113:591e600.
14] Vlodavsky Sachs L. Lectin receptors on the cell surface membrane and the
kinetics of lectin-induced cell agglutination. Exp Cell Res 1975;93:111e9.
15] Aicher D, Wiehe A, Stark C. Synthesis of glycoporphyrins using tri-
chloroacetimidates as glycosyl donors. Synlett 2010;2010:395e8.
16] Laville I, Figueiredo T, Loock B, Pigaglio S, Maillard P, Grierson D, et al. Syn-
thesis, cellular internalization and photodynamic activity of glucoconjugated
derivatives of tri and tetra(meta-hydroxyphenyl)chlorins. Bioorg Med Chem
[42] Durantini EN, Silber JJ. Synthesis of 5-(4-acetamidophenyl)-10,15,20-tris(4-
substituted phenyl)porphyrins using dipyrromethanes. Synth Commun
1999;29:3353e68.
[43] Monti D, Venanzi M, Mancini G, Marotti F, La Monica L, Boschi T. Synthesis and
complexation properties of an oligooxaethylene-spacered porphyrin dimer e
toward the construction of a new switchable porphyrin array. Eur J Org Chem
1999;1999:1901e6.
[44] Kuhne M, Gyorgydeak Z, Lindhorst TK. A simple method for the preparation of
glycosyl isothiocyanates. Synthesis 2006;6:949e51.
[45] Jimenez Blanco J, Sylla B, Ortiz Mellet C, Garcia Fernandez JM. Synthesis of alfa
and beta-glycosyl isothiocyanates via oxazoline intermediates. J Org Chem
2007;72:4547e50.
2003;11:1643e52.
[
17] Tomé JPC, Silva EMP, Pereira AMVM, Alonso CMA, Faustino MAF,
Neves MGPMS, et al. Synthesis of neutral and cationic tripyridylporphyrin-D-
galactose conjugates and the photoinactivation of HSV-1. Bioorg Med Chem
[46] Lindhorst TK, Kieburg C. Solvent-free preparation of glycosyl isothiocyanates.
Synthesis; 1995:1228e30.
2
007;15:4705e13.
18] Garcia G, Naud-Martin D, Carrez D, Croisy A, Maillard P. Microwave-mediated
click-chemistry” synthesis of glycoporphyrin derivatives and in vitro
[47] L’abbé G, Leurs S, Sannen I, Dehaen W. Synthesis of thiazolo [4,3-a]isoindoles
by intramolecular cycloaddition-elimination reactions of 4-methyl-5-
(substituted)imino-1,2,3,4-thiatriazolines. Tetrahedron 1993;49:4439e46.
[
“

80
J.C.C. Dallagnol et al. / Dyes and Pigments 107 (2014) 69e80
[
[
[
48] Falconer RA, Toth I. Design, synthesis and biological evaluation of novel lip-
oamino acid-based glycolipids for oral drug delivery. Bioorg Med Chem
[55] Sansone F, Chierici E, Casnati A, Ungaro R. Thiourea-linked upper rim calix[4]
arene neoglycoconjugates: synthesis, conformations and binding properties.
Org Biomol Chem 2003;1:1802e9.
[56] Paizs B, Pinter I, Csizmadia IG. An exploratory study of 1,2-cis- and 1,2-trans-
thiocarbamates of glucofuranosyl- and glucopyranosylamine. J Mol Struct
Theochem 1998;455:267e74.
[57] Plutín AM, Suárez M, Ochoa E, Machado T, Mocelo R, Concellón JM, et al.
Synthesis of new acyl, furoyl, and benzoylthiocarbamates as polydentate
systems. Structural study of isopropyl N-(2-furoyl)thiocarbamate. Tetrahe-
dron 2005;61:5812e7.
[58] Bautista-Sanchez A, Kasselouri A, Desroches MC, Blais J, Maillard P,
Oliveira DM, et al. Photophysical properties of glucoconjugated chlorins and
porphyrins and their association with cyclodextrins. J Photochem Photobiol B
Biol 2005;81:154e62.
2007;15:7012e20.
49] Prata C, Mora N, Lacombe JM, Maurizis JC, Pucci B. Synthesis and surface-
active properties of glycosyl carbamates and thioureas. Carbohydr Res
1999;321:4e14.
50] Zhou Y, Wang L, Han L, Meng F, Yang C. Synthesis, antifungal activities, and
potential detoxification of N-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)
thiocarbamates. Carbohydr Res 2009;344:1289e96.
[
51] Joshi UM, Patkar LN, Rajappa S. Concomitant desulfurization and trans-
esterification of alkyl thionocarbamates. Synth Commun 2004;34:33e9.
52] Viana GM, Aguiar LC de S, Ferrão J de A, Simas ABC, Vasconcelos MG. The use
of aqueous potassium dichloroiodate for the synthesis of ureas. Tetrahedron
Lett 2013;54:936e40.
[
[
[
53] Ichikawa Y, Nishiyama T, Isobe M. Stereospecific synthesis of urea-tethered
neoglycoconjugates starting from glucopyranosyl carbamates. Tetrahedron
[59] Kasha M, Rawls HR, El-Bayoumi MA. The exciton model in molecular spec-
troscopy. Pure Appl Chem 1965;11:371e92.
[60] Ali M, Pandey S. Aggregation behavior of aqueous tetrakis(4-sulfonatophenyl)
porphyrin in the presence of 1-butyl-3-methylimidazolium tetrafluoroborate.
J Photochem Photobiol A Chem 2009;207:288e96.
2004;60:2621e7.
54] Ichikawa Y, Ohara F, Kotsuki H, Nakano K. A new approach to the neo-
glycopeptides: synthesis of urea and carbamate-tethered N-acetyl-D-glucos-
amine amino acid conjugates. Org Lett 2006;8:5009e12.