Synthesis and photophysical evaluations of β-fused uracil-porphyrin derivatives

Article abstract of DOI:10.1016/j.tet.2013.09.069

New β-fused uracil-porphyrin conjugates were synthesized by the tetramerization of uracil-pyrroles under acidic conditions. Two different synthetic approaches were systematically studied in order to evaluate their efficiency, as well as the possibility to

Full text of DOI:10.1016/j.tet.2013.09.069

Accepted Manuscript
Synthesis and photophysical evaluations of β-fused Uracil-Porphyrin Derivatives
Carla Marisa Brito Carvalho, Marta Akemi Fujita, Timothy John Brocksom, Kleber
Thiago de Oliveira
PII:
S0040-4020(13)01491-9
10.1016/j.tet.2013.09.069
TET 24844
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 July 2013
Revised Date: 20 September 2013
Accepted Date: 23 September 2013
Please cite this article as: Brito Carvalho CM, Fujita MA, Brocksom TJ, Thiago de Oliveira K, Synthesis
and photophysical evaluations of β-fused Uracil-Porphyrin Derivatives, Tetrahedron (2013), doi:
10.1016/j.tet.2013.09.069.
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ACCEPTED MANUSCRIPT
Synthesis and photophysical evaluations of β-fused
Uracil-Porphyrin Derivatives
Carla Marisa Brito Carvalho, Marta Akemi Fujita, Timothy John Brocksom and
Kleber Thiago de Oliveira*
Departamento de Química, Universidade Federal de São Carlos, 13565-905, São Carlos - SP – Brazil.
Corresponding author Phone: +5516-3351-8083/+5516-3351-8213.
E-mail: kleber.oliveira@ufscar.br.
*
Homepage: http://www.lqbo.ufscar.br
Keywords: Porphyrin, uracyl, photodynamic therapy, singlet oxygen.
1

ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and photophysical evaluations of β-fused Uracil-
Porphyrin Derivatives
2

ACCEPTED MANUSCRIPT
Abstract
New β-fused uracil-porphyrin conjugates were synthesized by the tetramerization of uracil-
pyrroles under acidic conditions. Two different synthetic aproaches were systematically studied
in order to evaluate their efficiency, as well as the possibility to obtain a single regioisomer.
Metalation effects were studied for agreggation in solution, and preliminary photophysical
experiments were also performed in order to evaluate the potential of these new compounds.
3

ACCEPTED MANUSCRIPT
1
. Introduction
Porphyrin derivatives possess a well-established position in an extensive range of fields
1
2
3
4
such as catalysis, optical sensors, electronic devices, supramolecular systems, photovoltaic
5
6
7
cells, solar energy conversion and medicine. In this way, the synthetic community has
concentrated efforts on developing new compounds with better features for such applications.
A subject that has gained particular interest in the last 10 years involves the construction
of porphyrinic systems bearing uracil units. Drain et. al. designed and synthesized tetrameric
square supramolecules of meso-uracil-porphyrins that self-assemble by complementary hydrogen
8
bonds (Figure 1). The same authors also report that porphyrins bearing uracil motifs at the four
meso-positions self-organize via homo-complementary hydrogen bonds and π-stacking
interactions into nanofibers, nanorods and thin films on mica and glass surfaces, depending on
9
the deposition conditions. Takeoka and co-workers have synthesized an atrop-regulated
porphyrin derivative which bears 6-methyluracil units at the meso-position. Depending on the
addition of a melanine derivative, two assemblies were obtained, with different potential
1
0,11
applications as optical devices or catalysts for dioxygen reductions.
Renganathan et. al.
studied electron transfer processes of sulfonatophenylporphyrin to uracil derivatives, by using
steady state and time-resolved fluorescence quenching techniques. They found that the
1
2
porphyrins serve as electron donors and the uracil units as electron acceptors. Tao and co-
workers have described the synthesis of meso-uracil-porphyrins with very good properties and
1
3
photodynamic activities.
4

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O
O
N
H
H
N
N
N
N
HN
N
NH
N
N
N
H
NH
HN
O
O
N
O
O
N
H
O
O
N
N
H
N
H
H
N
H
N
H
N
N
O
O
O
O
N
O
O
N
N
N
H
H
O
N
O
NH
N
N
N
HN
N
H
HN
N
NH
Figure 1. Supramolecular assembly of a meso-uracil porphyrin.
Porphyrin systems bearing fused rings have been subject of intense research, since
they constitute a viable approach to obtain new compounds with improved photophysical and
1
4,15,16
photobiological properties.
Typically, the synthetic methodologies to obtain these compounds involve the direct
functionalization of the porphyrin core structure. However, this strategy has one disadvantage:
the β-pyrrolic positions have different reactivities which limit the scope and degree of
14
derivatization. In order to overcome this drawback, previous functionalization of the pyrrole
unit and then tetramerization, could constitute an important route to obtain new tetrapyrrolic
derivatives. The use of 3-, 4- or 3,4-substituted pyrroles with electron withdrawing groups for the
1
7,18
synthesis of the corresponding porphyrin derivatives is a challenging topic.
This is due to the
fact that porphyrinic macrocycles bearing electron withdrawing groups at the β-pyrrolic position
1
6,19,20,21
usually have interesting photophysical properties for different applications.
To the best of
5

ACCEPTED MANUSCRIPT
our knowledge, functionalization of porphyrins at the β-pyrrolic position with uracil units has not
been previously described. Thus, the preparation of these compounds represents a synthetic
challenge and the obvious building blocks are the substituted pyrroles bearing withdrawing
groups.
Herein we describe a study on tetra-β-uracil-porphyrin synthesis, starting from a
synthesized β-uracil-pyrrole and also a 2-hydroxymethylenic β-uracil-pyrrole derivative. We
have also performed metalation studies on the uracil-porphyrin, as well as structural and
photophysical characterizations. The aggregation phenomenon in solution was also assessed, and
we found no evidence of aggregation at the evaluated concentrations in solution.
2
. Results and Discussion
The required uracil-pyrrole 3 was prepared according to the synthetic strategy shown in
Scheme 1. Treatment of uracil (1) with K CO , followed by reaction with benzyl bromide
2
3
afforded the N-protected uracil 2. Subsequently, the reaction of this intermediate with
toluenesulfonylmethylisocyanide (TosMIC) in the presence of NaH in DMF/ethyl ether provided
2
2
the N-protected uracil-pyrrole 3.
Scheme 1: Synthesis of the uracil-pyrrole 3. i) K CO , DMF, r.t., 18h; ii) PhCH Br, rt., 72 h,
2
3
2
9
3% yield; iii) NaH, TosMIC, DMSO/Ether, r.t., 15h, 67% yield.
6

ACCEPTED MANUSCRIPT
The synthetic route for the novel porphyrin compound 6b is depicted in Scheme 2.
Initially, several attempts to tetramerize the pyrrole 3 were performed with different acids and
oxidation conditions (Table 1). The first evidence that β-uracil-porphyrin 6b can be obtained
from the acid-catalyzed tetramerization approach (route A), occurred when we performed the
reaction by condensation of uracil-pyrrole 3 with formaldehyde (from trioxane precursor) in the
presence of TFA at room temperature for 24 h, followed by oxidation with DDQ (Table 1, entry
2
3,24,25
7
).
7

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O
O
Bn
N
Bn
O
N
iv
NH
CH OH
NH
iii
3
O
N
N
CHO
Bn
2
Bn
4
5
A
B
i and ii
O
Bn
Bn
O
N
v and vi
Bn
N
R
M
R
O
N
N
O
Bn
N
N
N
N
R
R
Bn
O
N
N
O
Bn
N
N
Bn
O
Bn
O
6
a, R = Ph, M = 2H, not formed
6
b, R = H, M = 2H
viii
vii
6c, R = H, M = Zn(II)
d, R = H, M = Pd(II)
6
Scheme 2: Synthesis of the β-uracil-porphyrin 6. i) R-CHO, TFA, r.t., 24h; ii) DDQ, 1,2-DCE,
0ºC,48h, 8.5 % yield; iii) POCl , DMF, 1,2-DCE, 50ºC, 2h, 79% yield; iv) NaBH , THF, 0 ºC,
9
3
4
2
h, 89% yield; v) TFA, r.t., 2h; vi) DDQ, 1,2-DCE, 90ºC, 48h, 12 % yield; vii) Pd(OAc)2,
CH Cl /MeOH, 40ºC, 92% yield; viii) Zn(OAc) , CH Cl /MeOH, r.t., 95% yield.
2
2
2
2
2
8

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Table 1: Reaction conditions for the tetramerization of pyrrole 3.
Entry*
Pyrrole 3
ꢀmol)
R-CHO
(ꢀmol)
Conditions
Product
(%)
(
1
151.0
Ph (151.0)
Ph (151.0)
1. BF
3
.Et
2
O cat., CH
2
Cl
2
, 0 °C–reflux, 24h
2
. DDQ, 1,2-DCE, r.t. – 70 °C, 24h
1. BF .Et O cat., Zn(OAc)2, CH Cl , r.t.-reflux, 24 h
. DDQ or DDQ/NEt or p-cloranil or p-cloranil/NEt r.t., 24h
-
2
151.0
3
2
2
2
2
-
-
3
3,
3
4
151.0
151.0
Ph (151.0)
Ph (151.0)
Propionic acid, Nitrobenzene, 90-120 °C, 24h
1. TFA, r.t.-reflux, 24h
-
-
2
. DDQ, 1,2-DCE, r.t., 24h
5
**
151.0
H (151.0)
1. BF .Et O cat., 1,2-DCE, ꢀ, 24h
3
2
2
. DDQ, r.t., 3 days
6
7
151.0
151.0
H (151.0)
H (151.0)
Propionic acid, Nitrobenzene, 90-120 °C, 3 days
-
1. TFA, r.t. , 24h
Traces
2
. DDQ, 1,2-DCE, r.t., 24h
8
9
151.0
151.0
151.0
H (151.0)
H (151.0)
H (151.0)
1.TFA, 50 ºC , 24h
-
-
2
.DDQ, 1,2-DCE, r.t., 24 h
3.TFA, 85 ºC , 24h
.DDQ, 1,2-DCE, r.t., 24 h
4
1
0
1
2
3
4
5
6
1.TFA, r.t. , 24h
.DDQ, 1,2-DCE, r.t., 3days
1.1
2
1
151.0
302.0
302.0
302.0
302.0
302.0
H (151.0)
H (302.0)
H (302.0)
H (302.0)
H (302.0)
H (302.0)
1.TFA, r.t. , 24h
.DDQ, 1,2-DCE, r.t., 6days
1.4
2.6
2
1
1
1
1
1
1.TFA, r.t. , 24h
.DDQ, 1,2-DCE, r.t., 3 days
2
1.TFA, r.t. , 24h
.DDQ, 1,2-DCE, reflux, “Overnight”
4.2
2
1.TFA, r.t. , 24h
.DDQ, 1,2-DCE, reflux, 48 h
8.5
2
1.TFA, r.t. , 2h
.DDQ, 1,2-DCE, reflux, 48 h
Traces
-
2
1. TFA cat., r.t., 24 h
. DDQ, 1,2-DCE, reflux, 48 h
2
*
All reactions were tested in duplicate.
9

ACCEPTED MANUSCRIPT
*
* Not tested. BF . Et O was used to promote trioxane degradation.
3
2
cat.
The TLC analysis of the reaction revealed the formation of a new isolable compound,
with a pronounced green colour. Typical UV-Vis spectrum of the crude reaction revealed the
presence of a porphyrin derivative in the reaction mixture. After workup and purification, the
mass spectrum of the compound showed an m/z value at 1367 (from MALDI analysis)
corresponding to the expected macrocycle 6b. With these results in hands, and knowing that the
2
6
propionic acid/nitrobenzene methodology was not efficient for the preparation of the target
compound (Table 1, entry 6), we decided to test other conditions by using the TFA/DDQ
method.
One crucial parameter for the efficacy of porphyrin synthesis is the formation of the non-
oxidized intermediate, namely the porphyrinogen. In this way, we evaluated the influence of
temperature in this reaction step (Table 1, entry 8 and 9). However, no porphyrin was detected,
neither by TLC nor UV-Vis spectroscopy, when condensation of pyrrole 3 with formaldehyde
was conducted at 50 and 85 ºC. The influence of condensation time and quantity of acid (Table
1
, entry 15 and 16, respectively) was also evaluated but no significant amount of porphyrin was
observed in either case.
We have also studied the influence of time and temperature on the oxidation step (Table
, entry 10-14) and we find that both parameters are important for the success of the reaction.
1
Regarding the NMR characterization of compound 6b, some considerations have to be
1
taken into account. The H NMR spectrum of porphyrin 6b obtained from pyrrole 3 (Figure 2a)
was not in agreement with a pure compound. However, even after numerous preparative TLC
purification procedures with a wide range of solvents, TLC analysis revealed the presence of an
apparently single compound. Therefore, we speculated that an inseparable mixture of
1
0

ACCEPTED MANUSCRIPT
regioisomers had been generated. In order to solve this problem we decided to attempt an
alternative pathway, to obtain a single regioisomer (Scheme 2, route B).
For this purpose, the pyrrole-2-carboxaldehyde 4 was prepared in 79% yield according to
2
7
the Vilsmeier-Haack methodology. Analysis of the NOESY spectrum of 4 demonstrated the
regioselectivity of the reaction, since a correlation between the benzylic and formyl hydrogen
atoms was observed (see Supporting Information). Reduction of 4 with NaBH in THF provided
4
the 2-hydroxymethylenic-uracil-pyrrole 5 in 89 % yield. Several attempts to condense pyrrole 5
were carried out with different acid catalysts and oxidation conditions (Table 2). Initially, we
compared the reactivity of pyrroles 3 and 5 towards condensation in the same conditions (Table
1
, entry 14 and Table 2, entry 1). However, pyrrole 5 only produced traces of uracil-porphyrin 6b
in these conditions.
1
1

ACCEPTED MANUSCRIPT
Table 2: Formation of compound 6b using pyrrole 5 in different reaction conditions.
Entry*
Pyrrole 5
mol/ꢀmol)
Conditions
Product
(%)
(
1
1
38.4
38.4
1. TFA, r.t., 24h
. DDQ, 1,2-DCE, reflux, 48 h
1
2
Traces
12.2
3. TFA, r.t., 2h
. DDQ, 1,2-DCE, reflux, 48 h
2
3
4
5
6
4
276.8
138.4
138.4
138.4
1.TFA, r.t., 2h
.DDQ, 1,2-DCE, reflux, 48 h
2
12.1
2.1
1. TFA, 0 °C, 2 h
. DDQ, 1,2-DCE, reflux, 48 h
2
1. TFA, r.t., 2 h
. Nitrobenzene, reflux, 48 h
2
4.2
1. TFA cat., CH
Cl
2
, 2h, r.t.
2
1
1
4
2
.9
.7
.8
.1
2
. DDQ, 1,2-DCE, reflux, 48 h
7
8
9
138.4
138.4
138.4
1. TFA cat., CH
2
Cl
2
, 2h, r.t.
2
. NEt , DDQ, 1,2-DCE, reflux, 48 h
3
1. BF
. DDQ, 1,2-DCE, reflux, 48 h
1. BF .(Et O) cat., CH Cl , ꢀ, 24h
. NEt DDQ, 1,2-DCE, reflux, 48 h
.(Et O) cat., CH Cl , ꢀ, 24h
3
2 2 2
2
3
2
2
2
2
3
,
*
All reactions were tested in duplicate.
With these results in hands, we decided to test the use of milder conditions for the
synthesis of 6b. Thus, we assessed the importance of the porphyrinogen generation time (Table
, entry 2 and 3). The decrease on porphyrinogen generation time resulted in a significantly
better yield (12 %), indicating that the alcohol 5 is more reactive than the corresponding
2
1
2

ACCEPTED MANUSCRIPT
precursor 3. Consequently, we evaluated the influence of temperature, quantity and type of acid
on the porphyrinogen formation (Table 2, entry 4-9), but only low yields of 6b were obtained.
1
Concerning the H NMR spectrum of compound 6b obtained by this method (Figure 2b),
we observed that it was rather different from the one acquired with the porphyrin synthesized by
the first approach, wherein different regioisomers can be formed. These data indicate that we
obtained a mixture of regioisomers (Figure 2a) when the first synthetic method was used
(Scheme 2, route A) (Table 1), and a unique regioisomer by the second approach (Scheme 2,
route B, Figure 2b).
Ph
CH
2
-H
1
2.5
12.0
11.5
11.0
10.5
10.0
9.5
9.0
f1 (ppm)
8.5
8.0
7.5
7.0
6.5
6.0
5.5
1
Figure 2a – H NMR (400 MHz, in CDCl ) of compound 6b obtained from pyrrole 3.
3
1
3

ACCEPTED MANUSCRIPT
Ph
CH
2
-H
1
2.5
12.0
11.5
11.0
10.5
10.0
9.5
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
f1 (ppm)
1
Figure 2b – H NMR (400 MHz, in CDCl ) of compound 6b obtained from pyrrole 5.
3
After the preparation of the uracil-porphyrin 6b, we decided to deprotect the pyrrole 3 and
evaluate the possibility of tetramerization of compound 7. Deprotection was performed with
AlCl in toluene at 85 ºC and 1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (7) was obtained in
3
6
4% yield (Scheme 3). Several attempts were tested in order to condense the deprotected pyrrole
with formaldehyde and benzaldehyde, but in all cases only polymeric insoluble products were
7
obtained.
1
4

ACCEPTED MANUSCRIPT
Scheme 3: Attempts to synthesize porphyrin 8. i) AlCl , toluene, 85ºC, 1h, 64% yield; ii)
3
+
(
CHO) , H ; iii) [O]
3
Deprotection of porphyrin 6b was also tested in different conditions, but no reaction or
degradation were observed in all attempts.
Furthermore, the synthesis of β-fused uracil-porphyrins prompted us to explore the
facility of these new macrocycles to complex different metals such as Zn(II) or Pd(II), which
2
8,29
could potentiate different applications.
Porphyrin 6b was reacted with Zn(OAc) and
2
Pd(OAc) and the corresponding porphyrinic complexes 6c and 6d were obtained in 95% and
2
9
2% yields, respectively (Scheme 2).
In order to study the potential of compounds 6b-6d as photosensitizers as well as
fluorescent markers, some photophysical parameters were determined. The UV-Vis spectra of all
the porphyrins were registered at different concentrations (1-120 ꢁM). Free-base porphyrin 6b
showed a broad absorption band at 668 nm, an adequate UV-Vis region for PDT studies. The
Beer-Lambert Law was always obeyed in the studied concentration range (1-120ꢁM), and we
found no evidence of aggregation for all three new porphyrin derivatives 6b-6d (Figure 2).
1
5

ACCEPTED MANUSCRIPT
a)
1,4
1
,4
,2
1
Concentration
µM)
447
(
1,2
1
y = 114828x + 0,0618
R² = 0,9975
1
120
90
5
37
60
y = 8049,7x + 0,0572
R² = 0,9974
A
0,8
0,6
0,4
0,2
0
30
A
0,8
0,6
0,4
0,2
0
25
20
581
15
y = 9774x + 0,1053
R² = 0,9952
1
5
4
3
2
1
0
6
03
y = 7590,4x + 0,0314
R² = 0,9988
0,00E+00 2,00E-05 4,00E-05 6,00E-05 8,00E-05 1,00E-04 1,20E-04 668
y = 5261,5x + 0,0468
R² = 0,9963
327
377
427
477
527
577
627
677
Concentration (μM)
Wavelenght (nm)
b)
Concentration
µM)
3
,5
2
1
1
1
1
,8
,6
,4
,2
1
(
2
1
9
20
0
6
27
60
y = 17295x + 0,0426
R² = 0,9987
3
2
0
5
A
A
1,5
1
605
0
0
0
0
,8
,6
,4
,2
0
20
y = 8688,3x+ 0,003
R² = 0,9996
1
1
5
4
3
2
1
5
0
4
55
y = 263893x + 0,0043
R² = 0,9983
0,5
0
3
27
377
427
477
527
577
627
677
0,00E+00
5,00E-05
1,00E-04
1,50E-04
Concentration (μM)
Wavelenght(nm)
c)
2
1
,8
,6
,4
,2
1
1
,8
1
20
0
1
1
1
9
6
1,6
0
1
,4
,2
1
6
02
30
y = 14339x + 0,0279
R² = 0,9998
1
A
2
2
1
5
0
5
A
417
y = 58032x - 0,0342
R² = 0,9999
0,8
0,6
0,4
0,2
0
0,8
0,6
10
5
0
,4
,2
0
4
0
3
2
0,00E+00
5,00E-05
1,00E-04
327
377
427
477 527
Wavelenght (nm)
577
627
677
1
Concentration(μM)
Figure 2. UV-Vis spectra for compounds a) 6b (metal free); b) 6c (Zn-complex); and c) 6d (Pd-
complex).
Fluorescence emission spectra of free base 6b and Zn- and Pd-complexes 6c and 6d in
de-aerated toluene are shown in Figure 3.The spectra were recorded under identical conditions,
1
6

ACCEPTED MANUSCRIPT
including absorptivity at the excitation wavelength, reflecting the relative fluorescence emission
quantum yields of the porphyrins. The fluorescence emission data and quantum yields are
summarized in Table 3. The following equation was used to determine the quantum yields:
=
where F denotes the integral of the corrected fluorescence spectra, A is the absorbance at the
excitation wavelength, and n is the refractive index of the medium. The subscripts “s” and “r”
denote the sample and the reference compound, respectively.
1,00
0,75
0,50
0,25
0,00
Porphyrin 6b
absorbance
Porphyrin 6d
absorbance
1
,0
emission
emission
0,8
0,6
0,4
0,2
0,0
3
50 400 450 500 550 600 650 700 750 800 850 900
wavelength (nm)
3
50 400 450 500 550 600 650 700 750 800 850 900
wavelength (nm)
Porphyrin 6c
absorbance
emission
1
0
0
0
0
0
,0
,8
,6
,4
,2
,0
400
500
600
700
800
900
wavelength (nm)
Figure 3. Absorption (black line) and emission (dotted red line) spectra for porphyrins 6b-6d in
toluene.
1
7

ACCEPTED MANUSCRIPT
Table 3: The fluorescence quantum yields (Φ ) of porphyrins 6b-6d
f
Fluorescenceλmax [nm] r.t.
Compound
Φf
Q(0-0)
Q(0-1)
6
b
671
630
672
735
671
729
0.150
0.006
0.070
6
c
6
d
The ability of the macrocycles 6b-6d to generate singlet oxygen was qualitatively
estimated by measuring the absorption decay of 1,3-diphenylisobenzofuran (DPBF) at 410 nm
and compared with methylene blue (Figure 4 and Figures S24-27 in the Supporting Information).
Figure 4 – DPBF absorbance decay at 410 nm in the presence of different photosensitizers.
A considerable decay of 58 % was observed for the Pd(II) complex 6d and also for the Zn(II)
-2
derivative with 28 % decay, after 18 seconds of irradiation (60 mW.cm ). On the other hand, no
significant DPBF reduction (3%) was observed with the free-base 6b.
1
8

ACCEPTED MANUSCRIPT
It is important to note that no significant photobleaching decays were registered for all three
-2
porphyrins after 10 minutes of irradiation (60 mW.cm ) (see Figures S-28-30 in the Supporting
Information).
3
. Conclusion
In this work we have performed a comprehensive study on the synthesis of β-fused
uracil-porphyrins starting from two different pyrrole derivatives. We have demonstrated the
difficulties in synthesizing porphyrins bearing withdrawing groups at the β-positions, by
evaluating a number of methodologies. Another important aspect evidenced in these studies was
the difficulty to synthesize meso-arylporphyrins containing β-substituted pyrroles with hindered
and/or withdrawing groups. The new porphyrin derivatives 6b-d demonstrated their potential in
porphyrin chemistry, since were observed no aggregation at 1-120 ꢁM, good/moderate singlet
oxygen generation and fluorescence emission.
4
.
Experimental Section
4
.1 Chemicals.
All the reagents used were purchased from Sigma-Aldrich, Vetec (Brazil) or Synth (Brazil).
3
0
When necessary, solvents and reagents were purified using standard procedures.
4
.2 Physical measurements.
1
13
1
1
1
13
H NMR, C NMR and 2D experiments (gHSQC ( H/ H), gHMBC ( H/ C) and NOESY) were
performed on a Brüker AVANCE 400 operating at 400.15 MHz and 100.62 MHz, respectively.
1
9

ACCEPTED MANUSCRIPT
CDCl was used as solvent and tetramethylsilane (TMS) as internal reference. The chemical
3
shifts are presented in δ (ppm) and the coupling constants (J) in Hertz (Hz). High resolution mass
spectrometry (HRMS) spectra were recorded on Synapt G2-S, equipped with ESI and TOF.
4
.3 Photophysical measurements.
The UV-Vis spectra were recorded on Perkin Elmer Lambda 25 and fluorescence emission
spectra were recorded with Shimadzu RF-5301 . Singlet Oxygen measurements we performed
PC
by using a Varian Cary 50 spectrometer a laser Eagle Heron, 630 nm, 2W, Quantum Tech-São
Carlos-Brazil.
4
4
.2
Synthesis and characterization
.2.1 Synthesis of 1,3-dibenzylpyrimidine-2,4(1H,3H)-dione (2)
Anhydrous DMF (35 mL) was added through a septum to an argon-purged flask containing
uracil (1) (930 mg, 8.29 mmol) and K CO (2.75 g, 19.9 mmol). The mixture was stirred for 18
2
3
h, resulting in a thick gel. After this period, benzyl bromide (3.0 mL, 4.3 g, 25 mmol) was added,
and the reaction was stirred for 3 days. The reaction was quenched with water (50 mL) and
extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with water and
brine, dried over Na SO , filtered-off, and concentrated. Purification by column chromatography
2
4
on silica gel (1:1 EtOAc/hexane) afforded the product 2 in 93 % yield (2.25 g, 7.70 mmol), as
1
white crystals after crystallization from hexane/EtOAc. mp: 70-72ºC. H NMR (CDCl ,
3
4
00MHz) δ (ppm): 4.85 (s, 2H, CH -1), 5.11(s, 2H, CH -3), 5.68 (d, 1H, J = 7.9 Hz, H-5), 7.07
2 2 5,6
(
d, 1H, J = 7.9 Hz, H-6), 7.20-7.35 (m, 8H, H-p-3Bn, H-o-1Bn, H-m-3Bn, H-p-1Bn, H-m-1Bn,
5,6
2
0

ACCEPTED MANUSCRIPT
1
3
H-o-3Bn), 7.44-7.46 (m, 2H, H-o-3Bn). C NMR (CDCl ): δ (ppm): 44.4 (CH -3), 52.3 (CH -
3
2
2
1
), 102.1 (C-5), 127.6 (C-Ph), 128.0 (C-Ph), 128.4 (C-Ph), 128.5 (C-Ph), 128.9 (C-Ph), 129.1 (C-
Ph), 135.4 (C-1 ), 136.9 (C-3 ), 142.0 (C-6), 151.8(C-2), 162.9 (C-4). MS: m/z (relative
q
q
+
intensity), [M]· : 292 (28); 201 (16); 158 (62); 106 (40); 91 (100).
4
.2.2 Synthesis of 1,3-dibenzyl-1H-pyrrolo[3,4-d]pyrimidine-2,4(3H,6H)-dione (3)
Compound 2 (2.0 g, 6.8 mmol) was added to a slurry containing NaH 60% (0.82 g, 20.4 mmol)
o
in ethyl ether (30 mL) under a nitrogen atmosphere and at 0 C. After that, a solution of TosMIC
(1.99 g, 10.2 mmol) in ethyl ether:DMSO (4:1, 50 mL) was added dropwise. When the addition
was complete the resulting solution was stirred at r.t. for an additional 15 h. The reaction mixture
was quenched with a saturated solution of ammonium chloride (5 mL) and extracted with ethyl
ether (1 x 20 mL) and EtOAc (2 x 20 mL). The combined organic extracts were dried over
anhydrous Na SO , filtered-off and then concentrated under reduced pressure. Purification by
2
4
column chromatography on silica gel (1:1 EtOAc/hexanes) afforded the product 3 in 67% yield
(1.52 g, 4.58 mmol) as white crystals after crystallization from hexane/ EtOAc. mp: 167-169ºC.
1
H NMR (CDCl , 400MHz) δ (ppm): 5.05 (s, 2H, CH -7), 5.19 (s, 2H, CH -5), 6.49 (dd, 1H, H-2
3
2
2
1
1
13
or H-2 , J = 2.1, 0.4 Hz), 7.23-7.37 (H-Bn and H-2 or H-2 ), 9.90 (br.s., 1H, N-H). C NMR
1
(
CDCl , 100MHz) δ (ppm): 43.4 (CH -5), 48.1 (CH -7), 100.3 (C-2 or C-2 ), 105.2, 117.8 (C-2
3
2
2
1
or C-2 ), 126.5 (C-Ph), 126.8 (C-Ph), 127.0 (C-Ph), 127.6 (C-Ph), 128.0 (C-Ph), 128.2 (C-Ph),
+
1
36.6, 138.4, 151.6 (C6), 159.6 (C4). MS: m/z (relative intensity), [M]· 331 (19); 240 (4); 135
(33); 106 (20); 91 (100).
2
1

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4
.2.3 Synthesis of 1,3-dibenzyl-2,4-dioxo-2,3,4,6-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-
carbaldehyde (4)
o
A mixture of DMF (0.5 mL) and POCl (0.5 mL) was stirred under argon at 0 C for 5. After
3
warming to room temperature, it was stirred for an additional 30 min. An aliquot of this mixture
(335 ꢁL) was added to a solution of pyrrole 3 (500 mg, 1.51 mmol) in 1,2-dichloroethane (50
mL), warmed to 50 °C, stirred for an additional 2 h, cooled to room temperature, and slowly
poured into saturated aqueous NaHCO solution (50 mL) in an ice bath. The reaction mixture
3
was warmed up to room temperature, stirred for 10 min, then extracted with CH Cl (3x50 mL).
2
2
The organic layers were combined and washed with water (200 mL), dried over anhydrous
Na SO and evaporated under reduced pressure. The crude product was purified by column
2
4
chromatography on silica gel (hexane/EtOAc 1:1) to give the pyrrole 4 (420 mg, 1.19 mmol,
1
7
5
9%) as a white powder. mp: 253-256ºC. H NMR (CDCl , 400 MHz) δ (ppm): 5.13 (s, 2H, CH -
3
2
), 5.47 (s, 2H, CH -7), 7.18-7.36 (m, 10H, H-5-Bn and H-7-Bn), 7.93 (s, 1H, H-2), 9.36 (s, 1H,
2
1
3
H-10), 10.83 (br.s., 1H, N-H). C NMR (CDCl , 400MHz)δ (ppm):43.9 (C-5), 49.4 (C-7), 106.3
3
(C-3), 117.7 (C-9); 125.7, 126.8, 127.1, 127.2, 127.3, 128.3 and 128.8 (Ph);133.3 (C-8), 135.1
(
C -7), 137.5 (C -5), 151.6 (C-6), 158.4 (C-4), 177.5 (CHO). HRMS (ESI-TOF): m/z calculated
q
q
+
for [M+H] 360.1348; found 360.1360.
4
.2.4 Synthesis
of
1,3-dibenzyl-7-(hydroxymethyl)-1H-pyrrolo[3,4-d]pyrimidine-
2
,4(3H,6H)-dione (5)
2
2

ACCEPTED MANUSCRIPT
NaBH (40.0 mg, 1.06 mmol) was added to a solution of aldehyde 4 (125 mg, 0.35 mmol) in
4
THF (2.5 mL) at 0 ºC and stirred for 2 h at r.t. The reaction was quenched with water (25 mL)
and extracted with dichloromethane (4 x 15 mL). The organic layers were combined, dried over
anhydrous NaSO and evaporated under reduced pressure. The product was crystallized from
4
hexane/EtOAc to give pyrrole 5 (112.4 mg, 0.31 mmol, 89 %) as a white powder. mp: 177-
1
1
80ºC. H NMR (CDCl , 400 MHz ) δ (ppm): 4.17 (d, J = 4.8 Hz, 2H, CH -10), 5.11 (s, 2H,
3
2
CH -7), 5.25 (t, J = 4.8 Hz, 1H, O-H ), 5.29 (s, 2H, CH -5 ), 7.14 (d, J = 7.2 Hz, 1H, H-o-5 or 7-
2
2
1
3
Bn), 7.35 – 7.20 (m, 8H, H-5-Bn and H-7-Bn ), 7.45 (s, 1H, H-2), 11.93 (s, 1H, N-H). C NMR
(
CDCl , 100MHz) δ (ppm): 43.6 (C-5), 47.8 (C-7), 54.4 (C-10), 105.9 (C-3), 114.7 (C-9), 117.1
3
(
C-2), 125.5 (C-8 ); 127.2,127.4, 128.5 and 128.9 (Ph), 138.2 (C -7),138.4 (C -5),151.9 (C-
q
q
+
4
),159. 6 (C-6). HRMS (MALDI- TOF): m/z calculated for [M+H] 362.1505; found 362.1516.
4
.2.5 Synthesis of porphyrin 6b
TFA (6 mL) was added to a high pressure glass tube containing the uracil-pyrrole 5 (100.0 mg,
76.8 ꢁmol). The mixture was magnetically stirred at r.t. for 2 h. TFA was removed with a N2
2
flux and 1,2-dichloroethane (6 mL) and DDQ (70.0 mg, 310 ꢁmol) were added. The reaction
mixture was stirred at reflux for 48 h. The green porphyrin 6b was filtered on a sintered glass
funnel using preparative TLC silica and CH Cl /THF (9:1) as eluent. Finally, porphyrin 6b was
2
2
purified on preparative TLC using CH Cl /THF (99:1) as eluent and was obtained as a dark
2
2
1
green solid crystallized from CH Cl /MeOH in 12.1 % yield (11.4 mg, 8.37 µmol). HNMR
2
2
(
CDCl , 400MHz)δ (ppm): -5.57 (br.s, 1H, internal NH), -4.17 (br.s, 1H, internal NH), 5.53-5.83
3
(
m, 16H, CH ), 6.85-6.92, 7.30-7.67, 7.75-7.86 (3m, 40H, Ph),10.67-11.16 (m, 3H, meso-H),
2
2
3

ACCEPTED MANUSCRIPT
1
3
1
1
1
1.94 (br.s., 1H, meso-H). C NMR (CDCl , 100MHz) δ (ppm): 45.6, 49.8, 100.3, 102.3, 103.1,
3
04.1, 112.2, 126.6, 126.7, 127.3, 127.5, 127.6, 127.8, 127.9, 128.4, 128.5, 128.6, 129.2, 129.3,
29.5, 133.9, 134.8, 136.8, 136.9, 152.5, 159.7. HRMS (MALDI-TOF): m/z calculated for
+
[
(
M+H] 1367.4886; found 1367.4923. UV-vis (THF): λ (log ε): 447 (5.06); 537 (3.91); 581
max
3.99); 603 (3.88); 668 (3.72).
4
.2.6 Synthesis of complex 6c
Zn(OAc) (7.0 mg) was added to a solution of porphyrin 6b (5.0 mg; 3.5µmol) in
2
MeOH/CH Cl (1:2)(6mL). The reaction mixture was stirred for 24h at room temperature.
2
2
Product 6c was isolated after purification on preparative TLC using CH Cl /THF (99:1) as eluent
2
2
1
in 95% yield (5.0 mg, 3.49 µmol). H NMR (pyridine-d6, 400MHz) δ (ppm): 5.74-6.00 (m, 16H,
CH ), 6.96-7.10, 7.66-7.77, 7.99-8.12 (3m, 40H, Ph), 10.68(m, 1H,meso-H),10.78(s, 2H, meso-
2
+
H), 12.97 (s, 1H, meso-H). HRMS (MALDI- TOF): m/z calculated for [M] 1428.3943; found
1
428.3952. UV-Vis (THF): λmax (log ε) 455 (5.42); 605 (3.94); 627 (4.24).
4
.2.7 Synthesis of complex 6d
Porphyrin 6b (5.0 mg; 3.5µmol) was added to a saturated solution of Pd(OAc) in MeOH/CH Cl
2
2
2
(1:1) (1mL). The reaction mixture was stirred for 18 h at 40 ºC. Product 6d was isolated after
purification on preparative TLC using CH Cl /hexane (4:1) as eluent in 92% yield (4.7mg,
2
2
1
3
7
.22µmol). H NMR (pyridine-d6, 400MHz )δ (ppm): 6.01- 6.21 (m, 16H, CH ) , 6.91-7.01,
2
.41-7.91, 8.04-8.06 (3m, 40H, Ph), 10.10 (br.s., 1H,meso-H), 11.06-11.40 (m, 2H,meso-H),
2
4

ACCEPTED MANUSCRIPT
+
1
1
2.55 (br.s., 1H, meso-H). HRMS (MALDI- TOF): m/z calculated for [M+H] 1470.3712; found
470.3775. UV-Vis (THF): λmax (log ε) 471 (4.76); 564 (3.87, shoulder); 602 (4.16).
4
.2.8 Fluorescence measurements
3
1
Fluorescence quantum yields (Φ ) were measured by the method described by Parker and Rees.
f
Diluted solutions of each porphyrin in toluene with absorbance 0.02 at the excitation wavelength
(417 nm or 430 nm) were degassed by ultrasound, and protected from light before the
3
2
measurements. Tetraphenylporphyrin (TPP) was used as reference (Φ = 0.10).
f
4
.2.9 Singlet oxygen measurements
The singlet oxygen measurements were conducted using 1,3-diphenylisobenzofuran (DPBF) as
3
3
singlet oxygen quencher. A solution of DPBF (100 ꢁM) in ethanol was prepared. Solutions of
the porphyrins 6b-6d were prepared in the same solvent, with an absorbance of 0.4 at the Soret
band. The cuvette was filled with 1.0 mL of the porphyrin solution and 1.0 mL of the DPBF
solution. The absorbance was recorded and then the solution was irradiated for 3 second periods,
-2
using a red laser operating at 60 mW.cm and 630 nm. Ten irradiations were made, each one
followed by an absorbance measurement. Experiments were done in triplicate. The same
procedure was performed with standard methylene blue (MB).
4
.2.10 Photobleaching studies
2
5

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Solutions of the porphyrins 6b-6d in ethanol with an absorbance value around 1 were prepared,
-2
and irradiated for 1 min using a laser intensity of 60 mW.cm . The absorbance spectra were
recorded always after the irradiations (10 times), in order to observe the possible photobleaching
by reduction of the photosensitizer concentration.
Acknowledgements
The authors would like to thank the São Paulo Research Foundation (FAPESP) for fellowships
(2011/17177-5 and 2012/04964-1) and financial support (2011/13993-2 and 2013/065324) as
well as the CNPq and CAPES. Thanks are also due to Prof. Dr. Edson R. Filho and Dr. Douglas
Ferreira for the MALDI TOF-HRMS measurements, and the Bruker laboratory in Brazil for ESI-
TOF measurements. Also, we would like to thank the great opportunity of interaction with the
University of São Paulo through the NAP-CatSinQ (Research Core in Catalysis and Chemical
Synthesis).
Appendix. Supplementary data
Supplementary data associated with this article can be found in the online version, at doi:
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Supporting Information
Synthesis and photophysical evaluations of β-fused Uracil-
Porphyrin Derivatives
Carla Marisa Brito Carvalho, Marta Akemi Fujita, Timothy John Brocksom and Kleber Thiago
de Oliveira*
Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luís, Km
2
35 - SP-310, 13565-905,
São Carlos - SP – Brazil
*
kleber.oliveira@ufscar.br
Contents list
1
13
H and C NMR spectra for all the compounds..................................................................S1-S22
UV-Vis spectra of the singlet oxygen studies for all porphyrins........................................S23-S26
UV-Vis spectra of photobleaching studies for all porphyrins.............................................S27-S29
HRMS – MALDI TOF of the compounds 6b, 6c and 6d………………………………...S30-S38
1