Photophysical properties and photochemistry of a sulfanyl porphyrazine bearing isophthaloxybutyl substituents

Article abstract of DOI:10.1016/j.dyepig.2014.10.004

A magnesium porphyrazine possessing isophthaloxybutylsulfanyl substituents in the periphery was synthesized and subjected to various photophysical studies, including optical absorption and emission measurements. Moreover, synchronous fluorescence spectra were recorded and a contour three-dimensional map of the excitation-emission of the studied porphyrazine was obtained. The porphyrazine macrocycle exhibited interesting solvatochromic effects in many different solvents. Upon excitation with visible light, it generated singlet oxygen with a low quantum yield, therefore when it was encapsulated in liposomes it exhibited no photocytotoxicity in the in vitro study on human carcinoma LNCaP cell line.

Full text of DOI:10.1016/j.dyepig.2014.10.004

Dyes and Pigments 113 (2015) 702e708
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Photophysical properties and photochemistry of a sulfanyl
porphyrazine bearing isophthaloxybutyl substituents
,
Sebastian Lijewski a, Mateusz Gierszewski a b, Lukasz Sobotta c, Jaroslaw Piskorz c,
Paulina Kordas a, Malgorzata Kucinska d, Daniel Baranowski e, Zofia Gdaniec e,
,
, *
Marek Murias d, Jerzy Karolczak f g, Marek Sikorski b, Jadwiga Mielcarek c
,
,
*
Tomasz Goslinski a
a Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan, Poland
b Applied Photochemistry Lab, Adam Mickiewicz University in Poznan, Umultowska 89b, Poznan, Poland
c Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan, Poland
d Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30, Poznan, Poland
e Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, Poznan, Poland
f Faculty of Physics, Adam Mickiewicz University in Poznan, Umultowska 85, Poznan, Poland
g Centre of Ultrafast Laser Spectroscopy, Adam Mickiewicz University in Poznan, Umultowska 85, Poznan, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A magnesium porphyrazine possessing isophthaloxybutylsulfanyl substituents in the periphery was
synthesized and subjected to various photophysical studies, including optical absorption and emission
measurements. Moreover, synchronous fluorescence spectra were recorded and a contour three-
dimensional map of the excitation-emission of the studied porphyrazine was obtained. The porphyr-
azine macrocycle exhibited interesting solvatochromic effects in many different solvents. Upon excitation
with visible light, it generated singlet oxygen with a low quantum yield, therefore when it was encap-
sulated in liposomes it exhibited no photocytotoxicity in the in vitro study on human carcinoma LNCaP
cell line.
Received 1 August 2014
Received in revised form
3 October 2014
Accepted 6 October 2014
Available online 14 October 2014
Keywords:
Macrocyclization
Porphyrazine
Spectroscopy
Fluorescence
Absorption
© 2014 Elsevier Ltd. All rights reserved.
Singlet oxygen
1. Introduction
and dendrimeric architectures have shown potential for other
azaporphyrins [11,12].
Porphyrazines (Pzs) are synthetic analogues of naturally occur-
ring porphyrins. The Pz macrocycle consists of four pyrrole rings
linked together with azamethine groups [1]. Pzs show unique
spectrochemical properties and potential applications in technol-
ogy and medicine, especially as photosensitizers in photodynamic
therapy (PDT) [2e5]. Pzs with peripheral sulfanyl substituents
revealed enhanced solubilities and higher singlet oxygen genera-
tion yields [6e9]. The main limiting factors for Pzs application in
PDT are their poor solubility in water, tendency to form aggregates
and photochemical instability. These drawbacks may be overcome
by modifying Pzs periphery [1,10] or by their encapsulation in
various drug delivery systems, of which liposomal formulations
In this study we report the synthesis and photochemical char-
acterization of a novel sulfanyl magnesium(II) porphyrazine with
isophthaloxybutyl substituents.
2. Experimental section
2.1. Materials
2.1.1. 2,3-Bis[4-(3,5-dimethoxycarbonylphenoxy)butylsulfanyl]
maleonitrile (3)
Dimercaptomaleonitrile disodium salt (465 mg, 2.50 mmol) and
dimethyl 5-(4-bromobutoxy)isophthalate 2 (2.15 g, 6.25 mmol)
were dissolved in anhydrous methanol (50 mL). The reaction
mixture was stirred under reflux for 6 h. The solvent was evapo-
rated and the residual brown oil was chromatographed (dichloro-
methane: methanol, 50:1, v/v) to give 3 as yellow-brown oil (0.91 g;
* Corresponding authors. Tel.: þ48 61 854 66 31.
E-mail addresses: jmielcar@ump.edu.pl (J. Mielcarek), tomasz.goslinski@ump.
edu.pl (T. Goslinski).
http://dx.doi.org/10.1016/j.dyepig.2014.10.004
0143-7208/© 2014 Elsevier Ltd. All rights reserved.

S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
703
54% yield), which when refrigerated tends to solidify to yellow
are the absorbance of the sample and standard at an excitation
wavelength, respectively, nx e the solvent refractive index for the
sample, nst e the solvent refractive index for the standard, Fk e the
constant describing the instrumental factors, including geometry
and other parameters, FsFt is the value of fluorescence quantum
yield of the standard.
amorphous solid. M.p. ¼ 75e81 ꢀC; Rf (dichloromethane: meth-
anol, 50: 1, v/v) 0.56; UVeVis (dichloromethane) lmax nm (log )
3
318 (4.13), 343 (4.23); 1H NMR (500 MHz, pyridine-d5)
d 8.52 (s, 2H,
C40, ArH), 7.91 (s, 4H, C20, C60, ArH), 3.97 (t, 3J ¼ 6.0 Hz, 2H,
SCH2CH2CH2CH2), 3.87 (s, 12H, COOCH3), 3.31 (t, 3J ¼ 6.5 Hz, 4H,
SCH2), 1.92 (m, 4H, SCH2CH2CH2), 1.91 (m, 4H, SCH2CH2CH2); 13C
Synchronous fluorescence spectra (SFS) were collected by
simultaneous scanning using the excitation and emission mono-
chromators, in the range from 290 nm to 750 nm at Dl ¼ 10, 20, 30,
40, 60, 80, 100, and 120 nm. However, after the preliminary selec-
tion only the data collected for Dl ¼ 20 nm were discussed below. A
contour map of the emission-excitation of 4 was obtained in
acetonitrile by recording the emission spectra in the range from
350 nm to 750 nm using the excitation wavelengths from 300 nm to
400 nm, spaced by 5 nm intervals in the excitation domain.
Fluorescence lifetime measurements were made at the Centre
for Ultrafast Laser Spectroscopy in Poznan, with the respective
fluorescence lifetime spectrophotometer setup. Time-Correlated
Single Photon Counting (TCSPC) technique, previously described
in detail elsewhere [15], was applied. Spectra-Physics pico/femto-
second laser system was used as the source of exciting pulses. This
included a Tsunami Ti: sapphire laser, pumped with a BeamLok
2060 argon ion laser, which generated 1e2 ps pulses at a repetition
rate of about 82 MHz and average power of over 1 W, tunable in the
720e1000 nm range. The repetition rate of the excitation pulses
varied from 4 MHz to a single-shot by using a model 3980-2S pulse
selector. Second and third harmonics of the picosecond pulse ob-
tained on a GWU-23PS harmonic generator could be used for
excitation, giving greater flexibility in the choice of the excitation
wavelength. Elements of an Edinburgh Instruments FL900 system
were used in the optical and control components of the system. The
pulse timing and data processing systems employed a biased TAC
model TC 864 (Tenelec) and a R3809U-05 MCP-PMT emission de-
tector with thermoelectric cooling and appropriate preamplifiers
(Hamamatsu).
NMR (125 MHz, pyridine-d5)
d
166.5 (C ¼ 0), 159.9 (CH2eOeC, ArC),
132.8 (CeCO, ArC), 123.5, (ArC), 122.3 (CN), 120.5 (ArC), 113.5
(NCeCeS), 68.3 (OeCH2, Bu), 52.8 (COOCH3), 35.4 (SeCH2, Bu), 28.5
(SCH2CH2, Bu), 27.4 (SCH2CH2CH2, Bu); MS (ES pos) m/z 693
[MþNa]þ, 709 [M þ K]þ. MS (ES neg) m/z 705 [M þ Cl]-. Anal. Calc.
for C32H34N2O10S2: C, 57.30; H, 5.11; N, 4.18; S, 9.56. Found: C, 57.46;
H, 5.62; N, 4.20, S, 9.54.
2.1.2. 2,3,7,8,12,13,17,18eOctakis[4-(3,5-dibutoxycarbonylphenoxy)
butylthio]porphyrazinato magnesium(II) (4)
Magnesium turnings (11 mg, 0.45 mmol) and a small crystal of
iodine were refluxed in n-butanol (10 mL) for 4 h. After cooling to
room temperature, the reaction mixture was transferred using a
syringe to a flask containing maleonitrile 3 (233 mg, 0.34 mmol),
and was heated under reflux for 22 h. Next, the reaction mixture
was cooled to room temperature, filtered through Celite, which was
then washed with toluene. Solvents were evaporated in a rotary
evaporator, which resulted in a dark blue residue, and was chro-
matographed using silica gel (dichloromethane: methanol, 50: 1, v/
v) and reverse phase column chromatography (methanol, than
dichloromethane) to give 4 as dark blue film (111 mg; 37% yield). Rf
(n-hexane: ethyl acetate, 7: 3, v/v) 0.44; UVeVis (dichloromethane)
lmax nm (log
3
) 317 (4.77), 378 (4.89), 501 (4.17), 611 (4.45), 672
(4.94); 1H NMR (500 MHz, pyridine-d5)
d
8.50 (s, 8H, C40, ArH), 7.87
(s, 16H, C20, ArH), 4.59 (s, 16H, SCH2), 4.34 (t, 3J ¼ 6.5 Hz, 32H,
COOCH2), 4.15 (s, 16H, SCH2CH2CH2CH2), 2.35 (bs, 32H,
SCH2CH2CH2), 1.64 (m, 32H, COOCH2CH2), 1.35 (m, 32H,
COOCH2CH2CH2), 0.87 (t, 3J ¼ 7.5 Hz, 48H, COOCH2CH2CH2CH3); 13
C
NMR (125 MHz, pyridine-d5)
d
166.0 (C ¼ 0), 160.0 (CH2eOeC, ArC),
2.3. Singlet oxygen generation study
158.3 (N]C Ar), 141.6 (CeS, Ar), 133.0 (CeCO, ArC), 123.2 (ArC),
120.2 (ArC), 68.8 (ArO-CH2), 65.8 (COOCH2), 35.7 (SeCH2), 31.4,
29.3, 28.0, 19.9, 14.3 (CH3); MS (MALDI) m/z 3378 [M þ H]þ. HRMS
(ESI) Calc. for C176H233MgN8O40S8: 3378.4060, Found: [M þ H]þ
3378.4009. HPLC purity 96.22e100.00% (Supplementary Content).
A singlet oxygen generation assay was performed according to
the procedure described in detail by Sobotta et al. [16]. Irradiation
was performed at 671 nm according to the Q-band maximum
wavelength of 4 in DMF.
Further we used a Jobin Yvon-Spex Fluorolog 3-22 spectroflu-
orometer with H10,330B-75 NIR-PMT module to determine the
values of quantum yield of singlet oxygen generation of 4. Macro-
cycle was excited at 380 nm in acetonitrile in order to record
luminescence of singlet oxygen at 1270 nm.
2.2. UV/Vis measurements
All solutions containing 4 were prepared prior to their absor-
bance, steady-state fluorescence, and fluorescence excitation
measurements. UVeVis absorption spectra were recorded on a
JASCO V-650 spectrophotometer in the spectral range from 300 nm
to 800 nm, whereas the emission spectra (steady-state fluorescence
excitation and emission spectra, synchronous fluorescence spectra
and 3D fluorescence spectra) were recorded on a Jobin Yvon-Spex
Fluorolog 3-22 spectrofluorometer. Fluorescence quantum yields
were calculated using quinine sulphate in 0.05 M H2SO4 as a
reference for S2 / S0 emission (FsFt ¼ 0.546) [13] and using zinc
phthalocyanine (ZnPc) in DMF (FsFt ¼ 0.17) [14] for S1 / S0 emis-
sion. Fluorescence quantum yields were calculated according to the
equation below:
2.4. Liposome preparation
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and
L-a-phosphatidyl-DL glycerol (chicken egg, PG) were purchased
from Avanti Polar Lipidse INstruchemie (Delfizijl, Netherlands).
Liposomes with 4 were prepared by a thin-film hydration method
[17,18]. Appropriate amounts of the lipid solutions in chloroform
(25 mg/mL) and 4 (0.8 mg/mL) were placed in glass test tubes,
mixed and evaporated to dryness using a rotary evaporator. Films
formed on the bottom of the glass test tubes were dried overnight
in a vacuum at room temperature to evaporate any remaining
chloroform. Subsequently, the dried films were hydrated with
HEPES buffered saline solution (10 mM HEPES, N-(2-hydroxyethyl)
piperazine-N'-(2-ethanesulfonic acid), 140 mM NaCl, pH ¼ 7.4) and
dispersed by vortexing for 10 min using a Vortex Genie 2 digital.
Resulting liposome suspensions were passed 21 times through
polycarbonate membranes with a pore diameter of 100 nm, using a
Z
ꢀ
ꢁ
FX 1 ꢁ 10ꢁA
st
ðnXÞ2
ðnstÞ2
FF ¼ FsFt
Fk
(1)
Z
ꢀ
ꢁ
Fst 1 ꢁ 10ꢁA
X
here, !Fx is the area under the emission curve of the sample, !Fst is
the area under the emission curve of the standard, and Ax and Ast

704
S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
syringe extruder (Avanti Polar Lipids) to obtain unilamellar lipo-
somes with a uniform size distribution. The molar ratio of the final
liposome formulation was 4 (0.1): PG (2): POPC (8).
2.5. Biological studies
The biological activity of 4 was determined using the LNCaP
human cancer cell line (prostate carcinoma). The cell line was ob-
tained from the European Collection of Cells Cultures (ECACC, Sal-
isbury, UK). Cells were cultured in DMEM medium without phenol
red, supplemented with 10% FBS, 1% penicillin/streptomycin and 1%
L
-glutamine at 37 ꢀC, in a humidified atmosphere containing 5%
CO2.
The light source used for illumination was a high power LED
Fig. 1. Synthesis of compounds 2e4. Reagents and conditions: (i) Br(CH2)4Br, K2CO3,
DMF, 50 ꢀC, 20
[20]; (ii) (NC) (NaS)C]C(SNa) (CN), MeOH, reflux, h; (iii)
Mg(OC4H9)2, n-BuOH, reflux, 22 h.
h
6
MultiChip Emitter (60 high efficiency AlGaAs diode chips, Roithner
LaserTechnik GmbH, Vienna, Austria) with a light intensity of
2.2 0.5 mW/cm2 at 690 nm (irradiation dose after 20 min was
2.64 J/cm2). The plates were exposed to light at a distance of 8 cm.
The total spectral irradiance at the level of the cells was measured
using an RD 0.2/2 radiometer with a TD probe (Optel).
The cytotoxic effect of the tested compound was determined in
dark and light conditions. Cells were seeded in 96-well plates at the
density 2 ꢂ 104 cells per well and incubated overnight under cell
culture conditions. Then, the cells were washed twice with Phos-
phate Buffered Saline (PBS, Sigma Aldrich, St. Louis, MI, USA) and the
tested compound at concentrations of 0.08; 0.16; 0.34; 0.68; 1.35;
data for the singlet states of 4, including absorption maxima for
both the Soret and Q-bands (l1 and l2), emission maxima e noted
for S2 / S0 (lF2) and S1 / S0 (lF1), and the fluorescence quantum
yields (ФF). Two characteristic bands in the absorption spectra from
300 nm to 800 nm e the Soret band, located between 376 and
383 nm and the Q-band located between 664 nm and 681 nm were
observed, and their exact positions were found to be dependent on
the solvent used (see Table 1 for details). When analyzing the po-
sition of the Soret band in different solvents, it was concluded that
the solvent polarity affects this band only weakly, whereas the
changes noted in the Q-band were found to be more complex. In
protic solvents (alcohols), its maximum was located at
670e674 nm, while in aprotic solvents at 664e681 nm. It is note-
worthy, that the Q-band was found to be more intense than the
Soret band, as it has been previously observed for other porphyr-
azine and phthalocyanine derivatives [21e23]. Both absorption
2.70; 5.40 mM for 4 in a medium containing 2.5% Fetal Bovine Serum
(FBS, Sigma Aldrich, St. Louis, MI, USA) was added to each of the
wells. Free liposomes (PG(2): POPC(8)) were prepared as controls.
Cells were incubated for 24 h and were washed twice with PBS, and
fresh medium was added to each well. The plates were irradiated for
20 min and the cell viability was determined after 24 h.
The cell viability after the PDT treatment was evaluated by the
bands correspond to
p,p* electronic transitions. Table 1 presents
MTT assay [19]. Namely, 170
methylthiazolydiphenyl-tetrazolium bromide (Sigma Aldrich, St.
Louis, MI, USA) solution (20 L; 5 mg/mL PBS) in culture medium
mL of the reaction solution containing
the energy gap between the first and the second exited states. The
largest energy gap was obtained in 1,4-dioxane and the smallest in
dimethylformamide.
m
was added to each well. Then, the cells were incubated 2 h under
Fig. 3 presents the emission spectra recorded at different exci-
tation wavelengths (lexc ¼ 380 nm and 620 nm) and a synchronous
fluorescence spectrum (Dl ¼ 20 nm) of 4 in acetonitrile. Typically,
the porphyrazine was excited with two excitation wavelengths
(380 and 620 nm) in all solvents, upon which it emitted fluores-
cence. It was found that when the Pz 4 solution was excited at
380 nm, two fluorescence bands were observed, lF2 peaking be-
tween 417 and 489 nm, and lF1 peaking between 686 and 712 nm.
The exact positions of these bands depended on the solvent (see
cell culture conditions and the plates were centrifuged at 190 ꢂ g
for 3 min. Formazan crystals were dissolved by adding 200
mL
DMSO (POCh, Gliwice, Poland). The absorbance was measured at
570 nm with a plate reader (Biotek Instruments, Elx-800). Cell
viability was calculated as a percentage of the control. The results
are presented as the mean
experiments.
SD from two independent
3. Results and discussion
The first synthetic step was the alkylation reaction of 3-
hydroxyisophthalic acid dimethyl ester 1 with 1,4-dibromobutane
following an earlier reported procedure [20] (Fig. 1). Compound 2
was used in the subsequent reaction with dimercaptomaleonitrile
disodium salt, giving a novel maleonitrile derivative 3. Macro-
cyclization reaction using compound 3 led to the symmetrical
magnesium porphyrazine 4.
Novel compounds were characterized by UVeVis, MS and
various NMR techniques (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC)
(see Supplementary Content). Pz 4 was carefully purified by column
chromatography and further analyzed by HPLC. The detailed anal-
ysis of 1H-1H COSY spectra and 1H-13C HMBC longerange correla-
tions are presented in the Supplementary Content.
3.1. Spectroscopic studies
The absorption spectra of 4 in selected organic solvents were
Fig. 2. Absorption spectra of 4 in different organic solvents (acetonitrile, dichloro-
shown in Fig. 2. Table 1 presents spectroscopic and photophysical
methane, acetone dimethylformamide, ethyl acetate, methanol).

S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
705
Table 1
Spectroscopic and photophysical data for the singlet states of 4.
Solvent
l1/nm (Soret-band) l2/nm (Q-band) lF2/nm (S2 / S0) ФF ꢂ 104 (S2 / S0) lF1/nm (S1 / S0) ФF ꢂ 103 (S1 / S0)
D
E (S2 ꢁ S1)/cmꢁ1
cyclohexane
1,4-dioxane
ethyl acetate
dichloroethane
dichloromethane
dimethyl sulfoxide
dimethylformamide 383
acetone
acetonitrile
1-hexanol
1-pentanol
2-butanol
1-butanol
2-propanol
1-propanol
ethanol
381
376
378
378
378
380
672
681
666
656
674
672
667
664
666
674
672
672
673
674
673
671
670
462
489
422
461
458
436
421
453
417
423
424
422
421
426
441
426
427
5.4
4.8
4.2
1.2
1.1
3.1
3.6
6.2
52.1
9.2
7.6
8.1
7.5
7.0
7.3
6.9
5.6
689
712
689
691
697
691
686
686
687
690
691
690
690
691
690
690
692
5.4
3.2
9.7
2.1
2.4
4.5
5.4
1.2
9.8
6.2
7.9
7.4
8.5
8.2
7.9
7.4
6.7
11,370
11,910
11,440
11,210
11,620
11,430
11,120
11,260
11,300
11,550
11,430
11,430
11,530
11,550
11,530
11,480
11,460
380
380
379
380
380
379
379
379
379
379
methanol
Table 1). This short-wavelength emission band seems to corre-
spond to the S2 / S0 radiative transition, while the long-
wavelength band to the S1 / S0 radiative transition. Similarly, in
the synchronous fluorescence and 3D fluorescence spectra, two
emissions were observed. Notably, the long-wavelength emission
maximum had the same location in the spectra excited by both
380 nm and 620 nm radiations.
In conventional luminescence spectroscopy, the emission
spectrum is obtained by scanning the emission monochromator
over various emission wavelengths, lem, at a particular constant
excitation wavelength, lexc. The excitation spectrum is obtained by
scanning the excitation over various wavelengths, and keeping the
particular emission wavelength constant. Synchronous fluores-
cence spectrum (SFS) is obtained by simultaneously scanning both
the excitation and emission wavelengths, keeping a constant dif-
ference between them. The synchronous fluorescence intensity (Is)
depends on various factors, including the concentration of the
analyte in the sample (c), and may be expressed by the following
equation:
constant describing the instrumental factors, including geometry
and other parameters [24,25].
The SFS study was found to be particularly useful for acid-base
equilibria in the ground and excited singlet states of various
organic molecules, including lumichrome and quaternary stilba-
zolium salts [26,27]. Fig. 3 shows the two different bands in the SFS
of 4 in acetonitrile. Noteworthy, SFS spectral bands are narrower
than those in a conventional emission spectrum, with the two
bands being clearly separated. The band with the maximum at
about 397 nm seems to correspond to the S2 / S0 transition, while
the other at about 685 nm to the S1 / S0 transition in all of the
solvents used.
Similar results were obtained by Gan et al. [28] for a series of
octaphenyl-porphyrazine-magnesium salts, with the emission
from the S1 state in THF observed at about 650 nm with the life-
times of 1e3 ns, whereas the band emitting between 400 and
550 nm has been identified as the S2 / S0 emission. The
ФF of the
S2 emission was below 10ꢁ4, while its lifetimes were too short for
the nanosecond-resolution equipment. Interestingly, for a series of
demetalated sulfur porphyrazines with polyetherol groups Ehrlich
et al. [29] have reported dual fluorescence. Depending on the
excitation wavelength, they observed the presence of short-
wavelength emission coming from the S2 state and at the same
time the long-wavelength emission from S1. Moreover, the in-
tensity of S1 / S0 emission significantly decreased with an increase
of the number of sulfurpolyetherol groups. For porphyrazines with
eight sulfurpolyetherol groups upon excitation at 350 nm short-
wavelength emission was dominant and present at
lmax ¼ 409 nm, and accompanied by very weak long-wavelength
emission at lmax ¼ 745 nm. Porphyrazine with four sulfurpolye-
therol groups excited at about 340 nm revealed two emission bands
with maxima at about 424 nm and 823 nm. This observation was
explained by a decrease of the S1 emission accompanied by an in-
crease of the number of sulfur atoms, because the radiationless
Is ¼ KcbExðlexcÞEmðlexc þ DlÞ
(2)
Here, c e is the concentration of the analyte; Ex e intensity of the
excitation spectrum at lexc; Em e intensity of the emission spec-
trum at lexc
þ
Dl; b e the thickness of the sample; K e is the
conversion to the sulfur (n,p*) state was present [29].
The excitation-emission map of 4 in acetonitrile exhibits a
relatively intense broad band when the sample is excited between
300 and 325 nm and the emission monitored between 400 and
425 nm (Fig. 4). A weaker fluorescence appeared when the sample
was excited between 325 and 375 nm and the emission was
monitored between 380 and 480 nm. In addition, the second
emission band is located between 600 and 725 nm for the excita-
tion between 300 and 325 nm. As the extension of this study in
Fig. 5A, we present a comparison between the absorption and
excitation spectra of 4 in acetonitrile. The excitation spectra of 4
Fig. 3. Comparison between the synchronous fluorescence spectrum (Dl ¼ 20 nm)
and the emission spectra (lexc ¼ 380 and 620 nm) in acetonitrile for 4.

706
S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
Supplementary Content) a plot of the absorbance vs. concentra-
tion at
l
¼ 500 nm is presented. According to this data, two
different linear correlations were found, the first in the range of
concentrations from 1.37 ꢂ 10ꢁ6 M to 4.29 ꢂ 10ꢁ6 M with
R2 ¼ 0.996 and the second from 4.86 ꢂ 10ꢁ6 M to 1.83 ꢂ 10ꢁ5
M
with R2 ¼ 0.999. For lower concentrations only a monomeric form
was present. However, we believe that at higher concentrations, the
dimer (or even a higher aggregate) of 4 is present, apart from the
monomer. In Fig. S4A and S4B (see Supplementary Content) the
plots of absorbance vs. concentration of 4 at
670 nm, respectively, were included. Summarizing, in the same
l
¼ 380 nm and
range of concentrations of 4 two different linear correlations,
namely at
l
¼ 380 nm and 670 nm in the range of concentrations
from 1.37 ꢂ 10ꢁ6 M to 4.29 ꢂ 10ꢁ6 M with R2 ¼ 0.998 and the
second form 4.86 ꢂ 10ꢁ6 M to 1.83 ꢂ 10ꢁ5 M with R2 ¼ 0.999, were
found. Further, the fluorescence excitation spectrum at
lem ¼ 430 nm was recorded. In Fig. 5B the excitation spectra of 4 at
concentrations equal to 1.37 ꢂ 10ꢁ6 M and 1.83 ꢂ 10ꢁ5 M recorded
at lem ¼ 430 nm were presented. The fluorescence excitation
spectra were compared to the absorption spectrum of 4 in the
Soret-band region. Generally, we noted satisfactory agreement
between the excitation spectrum and the Soret band in the ab-
sorption spectrum, which particularly match each other at ca.
380 nm.
Fig. 4. Excitation-emission contour map of 4 in acetonitrile.
was recorded at 430 nm and 750 nm as emission wavelengths
appeared at concentrations from the range 1.37 ꢂ 10ꢁ6 M and
1.83 ꢂ 10ꢁ5 M. The corresponding spectra of fluorescence excitation
and absorption match each other. The only exception appears in the
concentration of 1.83 ꢂ 10ꢁ5 M at about 600 nm, where some
changes in the relative band intensities are observed. We believe
that it might be due to the presence of weakly emitted dimers of 4.
The indications that dimers may be formed in solutions have been
found for some azaphthalocyanines [30]. In Fig. S4C (see
The fluorescence quantum yields (ФF) were calculated using the
procedure described in the experimental part. The data for the S2
emission are listed in Table 1 in selected organic solvents. Sum-
marizing, the S2 emission was found to be rather weak in all of the
solvents used, with quantum yields of only about 10ꢁ4, except for 4
in acetonitrile, where the quantum yield achieves 0.005. In addi-
tion, the fluorescence quantum yields for S1 emission are pre-
sented. The values of ФF (S1 / S0) are rather low, at about 10ꢁ3
,
with the highest value determined in acetonitrile, and the lowest in
acetone. The low values of fluorescence quantum yields might be
explained by the presence of eight sulfur atoms in 4, responsible for
heavy-atom deactivation of the excited states. Notably, the rela-
tively low fluorescence quantum yields were also found for zinc(II)
phthalocyanine functionalized by eight thioglucose units [31].
Similarly, the influence of sulfur present in the phthalocyanine ring
has been observed for octaglucosylated zinc(II) phthalocyanines,
containing oxygen or sulfur bridges. In that case, fluorescence
quantum yields were lower in derivatives containing sulfur bridges,
than the oxygen bridged analogue [32].
In addition, the fluorescence lifetimes for S1 emission in meth-
anol and acetonitrile and for S2 emission in acetonitrile were
measured. In acetonitrile, the excitation at 380 nm was used and
emission monitored at 427 nm. Mono-exponential fluorescence
decay was observed with tF (S2 / S0) ¼ 3 ps. As IRF (Instrument
Response Function) was used xanthione in acetonitrile (tR ¼ 12 ps,
lem ¼ 460 nm) [33]. Such a short lifetime can be attributed to the S2
state of 4. Moreover, the fluorescence decay of 4 in acetonitrile at
lexc ¼ 380 nm and lem ¼ 690 nm was accompanied by bi-
exponential decay with tF1
¼ 32.4 ps (40%) and tF2
(S1
/ S0)
¼ 105.6 ps (60%). Similarly, in methanol bi-exponential
(S1 / S0)
decay with tF1
¼
38.5 ps (40%) and tF2
(S1
/
S0)
¼ 116.8 ps (60%) was noted. Literature data indicates for
(S1 / S0)
some phthalocyanines and porphyrazines mono-exponential decay
of the S1 state on the ns time scale [31,32]. However, bi-exponential
fluorescence decay was also noted for porphyrazines with periph-
eral 2,5-dimethylpyrrol-1-yl and dimethylamino groups dissolved
in DMSO. The exact values of the fluorescence lifetimes were
1.24 ns (13%) and 2.95 ns (87%) for porphyrazine that was metalated
with Mg ion. The origin of the shorter component can be a result of
interaction between solvent (DMSO) and the metal cation at the
centre of the macrocycle [16]. The data collected in Fig. S4 (see
Fig. 5. Normalized absorption and excitation spectra A: lem
¼
750 nm and B:
lem ¼ 430 nm in acetonitrile for 4 at concentration 1.37 ꢂ 10ꢁ6 M and 1.83 ꢂ 10ꢁ5 M.

S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
707
Supplementary Content) and the decay contributions (in both cases
40%), indicate that the short-living species observed at
lem ¼ 690 nm are the dimers. The fluorescence lifetimes were
measured at about 4 ꢂ 10ꢁ6 M (in acetonitrile) and at about
4.5 ꢂ 10ꢁ6 M (in methanol).
3.2. Solvatochromic studies
The
the solvent polarity on the spectral and photophysical properties of
4. The f solvent polarity scale is based on the Onsager's reaction
Df solvent polarity scale was used to study the influence of
D
field theory and includes general solvent effects, including non-
specific interactions between solutes and solvents of electrostatic
and dispersive origin, arising from solvents acting as a dielectric
continuum and specific solute e solvent interactions such as
hydrogen bonding. The function describing the
scale is based on Lippert-Mataga solvent polarity function [34,35]:
Df solvent polarity
Fig. 6. Absorption spectra of a mixture of DPBF and 4 in DMF, during irradiation at
417 nm.
3
ꢁ 1
n2 ꢁ 1
Df ¼
ꢁ
(3)
2n2 þ 1
2
3
þ 1
Solvatochromic studies for 4 in different solvents using various
solvent polarity scales show that the correlations are rather poor.
For such a large molecule as 4 with different groups, which are
involved in specific and non-specific interactions, it is difficult to
obtain satisfactory correlations (even when using various solvent
polarity scales which include many types of interactions between
solvents and solutes). Better correlations (R2 ¼ 0.925) were ob-
tained in our previous study for 12 solvents when another phtha-
locyanine derivative bearing two 1-adamantylsulfanyl groups at
peripheral positions was applied. At that time, we considered only
the function of solvent refractive indices vs. Q-band maxima [37].
The Df parameter is accurately determined for most of the sol-
vents and is dependent on the dielectric constant
3
and the
refractive index n.
A more detailed analysis was performed using seventeen
different solvents, including cyclohexane, 1,4-dioxane, ethyl ace-
tate, dichloroethane, dichloromethane, dimethyl sulfoxide, dime-
thylformamide, acetone, acetonitrile, 1-hexanol, 1-pentanol, 2-
butanol, 1-butanol, 2-propanol, 1-propanol, ethanol and meth-
anol. However, this analysis did not provide useful correlations
(plots not shown). Using the regression of the nabs (Q-band) vs. Df,
the correlation coefficient R ¼ 0.33 was obtained for all solvents;
separately, R ¼ 0.56 was obtained for protic solvents (alcohols) and
3.3. Singlet oxygen generation study and in vitro photodynamic
activity results
R ¼ 0.65 for aprotic solvents. Using the regression of nem (in cmꢁ1
)
vs. the
D
f, R ¼ 0.57 was obtained for all solvents, with slightly better
correlations for the two separate groups of protic and aprotic sol-
vents, R ¼ 0.61 and 0.71, respectively.
The porphyrazine 4 generated singlet oxygen in DMF with a
quantum yield of 0.02, with no significant photodegradation during
Additionally for 4, we used the four-parameter scale proposed
the experiment (1.30
0.01%). The absorption spectra indicating
ꢀ
by Catalan [36], see Table S3 in Supplementary Content. The four-
sensitized photooxidation of diphenylisobenzofurane (DPBF) are
shown in Fig. 6.
ꢀ
parameter Catalan scale describes separately specific and non-
ꢀ
specific interactions between solvents and solutes. The Catalan
To determine the cytotoxicity of the tested compound, the MTT
assay for LNCaP prostate human cancer cell line was performed.
The results show no cytotoxic potential of 4 in liposomal formu-
lation. Moreover, a proliferative effect was observed after the PDT
experiment, probably of hormetic nature.
Singlet oxygen luminescence photosensitized by 4 was observed
in acetonitrile and dichloromethane, recording its characteristic
spectra with a maximum at about 1270 nm. Measurements were
done relative to the spectra of singlet oxygen obtained in acetoni-
trile and dichloromethane with perinaphtenone (ФD ¼ 0.95 0.05)
[38] as a standard for excitation at 380 nm. Methylene blue was
parallel utilized as a standard for excitation at 660 nm (ФD ¼ 0.57 in
dichloromethane and ФD ¼ 0.52 in acetonitrile) [39,40]. In both
solvents, acetonitrile and dichloromethane, the values of the
quantum yield of singlet oxygen generated by 4 excited at 380 nm
and 660 nm were 0.04 and 0.06, respectively.
scale is based on specific and general scales, using four parameters
(SA e solvent acidity, SB e solvent basicity, SdP e solvent dipolarity
and SP e solvent polarizability scales) [36].
A ¼ A0 þ aSPSP þ bSdPSdP þ cSASA þ dSBSB
(4)
Here, A0 is the statistical quantity corresponding to the value of the
property in the gas phase; SP, SdP, SA, and SB, represent solvent
parameters, and aSP, bSdP, cSA, and dSB are the regression coefficients
(solvent-independent), describing the sensitivity of the property A
to the different soluteesolvent interaction mechanisms [36]. In
Table S3 (Supplementary Content) results of the regression calcu-
lations for the multilinear regression analysis of the absorption and
emission maxima (in cmꢁ1) were presented. The parameter values
of the gas phase (A0) and the estimated coefficients: aSP, bSdP, cSA
,
and dSB with the correlation coefficient (R) were included. To
summarize, the absorption maxima are mostly affected by basicity
of solvents and solvent dipolarity with the R ¼ 0.81. A less
acceptable correlation coefficient (R ¼ 0.66) was obtained when all
4. Conclusions
ꢀ
of the Catalan parameters (solvent acidity, basicity, dipolarity and
A novel magnesium porphyrazine macrocycle was characterized
in terms of its spectrochemical properties. Detailed emission
studies were carried out, and its results permitted drawing a three
dimensional map of excitation-emission properties. Porphyrazine
exhibited interesting solvatochromic effects in the range of
different solvents. Interestingly, the macrocycle generated singlet
polariazability) were considered. For the emission maxima, a less
satisfactory correlation as compared to the absorption (R ¼ 0.73) for
ꢀ
three Catalan parameters: solvent polarizability, acidity and basic-
ꢀ
ity, was obtained. Noteworthy, for all Catalan parameters R ¼ 0.64,
was found.

708
S. Lijewski et al. / Dyes and Pigments 113 (2015) 702e708
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Acknowledgments
This study was supported by the Polish National Science Centre
under Grant No. 2012/05/E/NZ7/01204 and the European Fund for
Regional Development No. UDA-POIG.02.01.00-30-182/09. Pico-
second fluorescence decay measurements were made at the Center
for Ultrafast Laser Spectroscopy at the Adam Mickiewicz University
in Poznan, Poland.
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