Spectroscopic probing of the acid-base properties and photosensitization of a fluorinated phthalocyanine in organic solutions and liposomes

Article abstract of DOI:10.1562/0031-8655(2001)0730473spotab2.0.co2

A perfluorinated derivative of phthalocyanine was synthesized as the free base, hexadeca-(2,2,2-trifluoroethoxy) phthalocyanine (H₂F₄₈Pc), and as a zinc complex, hexadeca-(2,2,2-trifluoroethoxy)-phthalocyaninatozinc (ZnF₄₈

Full text of DOI:10.1562/0031-8655(2001)0730473spotab2.0.co2

Photochemistry and Photobiology, 2001, 73(5): 473–481
Spectroscopic Probing of the Acid–Base Properties and
Photosensitization of a Fluorinated Phthalocyanine in Organic Solutions
and Liposomes¶
Hana Weitman1, Smadar Schatz1, Hugo E. Gottlieb2, Nagao Kobayashi3 and Benjamin Ehrenberg*‡1
1
2
Departments of Physics and Chemistry, Bar Ilan University, Ramat Gan, Israel and
3Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Japan
Received 7 December 2000; accepted 17 February 2001
ABSTRACT
INTRODUCTION
A perfluorinated derivative of phthalocyanine was syn-
thesized as the free base, hexadeca-(2,2,2-trifluoroethoxy)
phthalocyanine (H2F48Pc), and as a zinc complex, hex-
adeca-(2,2,2-trifluoroethoxy)-phthalocyaninatozinc
(ZnF48Pc), and their spectroscopic and photochemical
properties were studied. The absorption bands are shift-
ed bathochromically relative to simple phthalocyanines,
exhibiting the longest wavelength band near 735 nm
(H2F48Pc) and 705 (ZnF48Pc). The solvatochromism of
both compounds was modeled by Reichardt’s ET(30) pa-
rameter and Kamlet, Abboud and Taft multiparameter
approach. The former, simpler, model was found to be
adequate. We found that H2F48Pc undergoes unique basic
and acidic titrations in organic solvents. These titration
processes are accompanied by spectral changes that are
explained on the basis of the chromophore’s symmetry.
Singular value decomposition was employed to resolve
the spectra into the contributions of the species at various
stages of protonation and to obtain the equilibrium con-
stants. Nuclear magnetic resonance spectra (1H, 19F and
13C) for the free base were obtained in a tetrahydrofuran-
d8 solution. The carbon spectrum, taken as a function of
temperature, provided evidence for the presence of a tau-
tomerization process, which switches the two internal hy-
drogens between the four central nitrogen atoms. As far
as we know, this is the first report of the measurement
Porphyrins are very important natural and synthetic com-
pounds. In recent years there has been a growing interest in
porphyrins and phthalocyanines due to their capacity to act
as photosensitizers and due to the application of photosen-
sitization to photodynamic therapy of malignant tumors. Al-
though the various properties of synthetic porphyrins have
been thoroughly studied very few reports of their basicities
or acidities in nonaqueous media have appeared so far and
even fewer for phthalocyanines (1,2).
In this paper we report the spectroscopic and photophysical
properties of the free base, hexadeca-(2,2,2-trifluoroethoxy)
phthalocyanine (H2F48Pc)† and the hexadeca-(2,2,2-trifluoro-
ϩ
ethoxy)-phthalocyaninatozinc Zn2 complex (ZnF48Pc) of a
highly fluorinated phthalocyanine (Fig. 1). The perfluorination
of the phthalocyanine core is expected to be responsible for
the lability of the core hydrogens by increasing their acidity,
which should enable the acid–base titration of the pyrrole ni-
trogens, even in organic solvents.
MATERIALS AND METHODS
Chemicals.
L-␣-phosphatidylcholine (L-␣-lecithin) from egg yolk
(ϳ
99%), 9,10-dimethylanthracene (DMA), hematoporphyrin IX di-
hydrochloride (HP) and hexanol were purchased from Sigma Chem-
ical Co. (St. Louis, MO). Spectrophotometric grade trifluoroacetic
acid (99
(5,6,11,12-tetraphenylnaphthacene) and meso-tetraphenylporphine
(99 %) (TPP) were obtained from Aldrich Chemical Co. (Milwau-
ϩ%) (TFA), N,N-dimethylformamide (DMF), rubrene
ϩ
of the free energy of activation for such process (⌬ ؍
G†
kee, WI). Methanol, hexane, acetonitrile and tetrahydrofuran (THF)
were obtained from Bio-Lab Ltd. Laboratories (Jerusalem, Israel).
Triethylamine (TEA), chloroform, dioxane, decanol, pyridine and
benzene were obtained from Merck (Darmstadt, Germany). Acetone
was obtained from Pharmco Products (Brookfield, CT). Ethanol was
obtained from Frutarom Ltd. (Haifa, Israel). Nitrobenzene was from
Fluka Chemie (Buchs, Switzerland).
Ϫ
10.6–11.4 kcal mol 1 between 217 and 330 K) for a phtha-
locyanine, in solution. Like most other phthalocyanines
these two compounds also act as photosensitizers and as
generators of singlet molecular oxygen. The absolute
quantum yields (
⌽
⌬) for ZnF48Pc was 0.58
؎ 0.01 in ben-
Synthesis of 3,4,5,6-tetrakis-(2Ј,2Ј,2Ј-trifluoroethoxy)phthalonitrile.
zene and 0.35
؎
0.01 in lipid vesicles. H2F48Pc had lower
yields, 0.16 and 0.005, respectively. Either protonation or
deprotonation of the pyrrole nitrogens in H2F48Pc low-
†Abbreviations: DMA, 9,10-dimethylantracene; DMF, N,N-dime-
thylformamide; DMSO, dimethylsulfoxide; H2F48Pc, hexadeca-
(2,2,2-trifluoroethoxy) phthalocyanine; HP, hematoporphyrin IX;
KAT, Kamlet, Abboud and Taft solvatochromism model; NMR,
nuclear magnetic resonance; PCA, principal component analysis;
rubrene, 5,6,11,12-tetraphenylnaphthacene; TEA, triethylamine;
TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMS, trimethyl-
silane; TPP, meso-tetraphenylporphine; SVD, singular value de-
composition; ZnF48Pc, hexadeca-(2,2,2-trifluoroethoxy)-phthalo-
cyaninatozinc.
ered the
⌽ .
⌬
¶Posted on the website on 28 February 2001.
*To whom correspondence should be addressed at: Department of
Physics, Bar Ilan University, Ramat Gan 52900, Israel. Fax: 972-
3-5353298; e-mail: ehren@mail.biu.ac.il
‡Incumbent of the David Falk Chair in laser phototherapy.
᭧
2001 American Society for Photobiology 0031-8655/01
$5.00ϩ0.00
473

474 Hana Weitman et al.
of the solvent under reduced pressure the residue was chromato-
graphed, using basic alumina and methylene chloride–hexane (4:1
vol/vol), methylene chloride and finally acetone–hexane (1:4 vol/
vol). After removing the solvent the green residue was recrystallized
from acetone–hexane to give 0.531 g (52%) of the desired ZnF48Pc.
Anal. Found: C, 36.02; H, 1.52; N, 5.09. Calc. for
C64H32N8O16F48Zn: C, 35.82; H, 1.50; N, 5.22%. 1H NMR (CD3CN,
TMS):
Liposomes preparation. A chloroform–methanol solution of
phosphatidylcholine and 30 L of ZnF48Pc or H2F48Pc stock solu-
␦ 5.653 (q, 16H), 5.035 (q, 16H).
L-␣-
␮
tions in THF (1 mM) was evaporated with N2 and the dry material
redissolved in diethyl ether, This, too, was evaporated. Water was
1
added to form a lipid concentration of 1 mg mLϪ and sonicated
with a probe sonicator (MSE Soniprep 150, Crawley, West Sussex,
UK) for 5 min
ϫ 3 at 4ЊC.
NMR measurements. 19F NMR and 400 MHz 1H spectra were
recorded with a JEOL GX-400 spectrometer using CDCl3, DMSO-
d6, CD3CN or their mixture. For the tautomerism study NMR spectra
1
were taken in THF-d8 on Bruker spectrometers: DMX-600 for H
and 13C (600.1 and 150.9 MHz, respectively) and AC-200 for 19F
1
(188.3 MHz). The data obtained (13C and H spectra are referred to
internal TMS, 19F to external CFCl3) are summarized in Table 4.
Probe temperatures were measured with a calibrated Eurotherm 840/
T digital thermometer, connected to a thermocouple, which was in-
serted into a liquid-filled NMR tube. The temperatures are accurate
to 0.5ЊC.
Figure 1. Molecular structure of H2F48Pc and ZnF48Pc.
Spectroscopic measurements and irradiations. Absorption spectra
were measured by a Perkin–Elmer Lambda 9 UV–Vis–near-IR com-
puter-controlled spectrophotometer. Fluorescence emission and ex-
citation spectra and fluorescence time-drive measurements were per-
formed on a digital computer-controlled fluorimeter (model LS-50B;
Perkin–Elmer, Norwalk, CT). IR measurements were made with Shi-
madzu FTIR-8100M spectrometer using KBr discs.
For the measurements of singlet oxygen quantum yield laser ir-
radiation of the sample was carried out in situ in the fluorimeter. A
He–Ne laser (model 1135; Uniphase, Stevenage, Hertfordshire, UK)
was used as the irradiation source for H2F48Pc and ZnF48Pc. For
irradiation of TPP and HP an Arϩ laser (model Innova 200; Coher-
ent, Palo Alto, CA) was used. The laser light’s power was measured
at the sample surface with a laser power meter (model PD2-A;
Ophir, Israel). The sample solution contained the sensitizer (at var-
2,2,2-Trifluoroethanol (8.0 g, 110.0 mmol) was added, under nitrogen
atmosphere, to a cooled (0ЊC) mixture of 60% NaH (4.40 g, 111.0
mmol) and THF (100 mL), placed in a three-necked flask (300 mL)
for 30 min, and the mixture was reacted at this temperature for 30
min. To this solution was added 5 g (25.0 mmol) of 3,4,5,6-tetrafluo-
rophthalonitrile, dissolved in 20 mL THF, over 30 min. The ice-water
bath was removed, and after warming up to room temperature, the
stirring was continued for another 30 min, and then 2 N hydrochloric
acid was poured into the mixture to stop the reaction. Water and
THF layers were identified. The THF layer was collected by decan-
tation, the solvent was removed by a rotary evaporator and the res-
idue was recrystallized from THF–hexane. The above-mentioned
water layer was extracted twice with ether, this ether solution was
washed with brine and dried over MgSO4. After removal of MgSO4
by filtration, the ether was evaporated, and the residue was crystal-
lized from THF–hexane. The crystallized product was chromato-
graphed on silica gel (Wako gel C-300) with methylene chloride–
hexane (2:1 vol/vol), and recrystallized from THF–hexane to yield
ious concentrations in the 3–10 ␮M range) and DMA (5 ␮M) which
was used as a chemical target for singlet oxygen, and was stirred
magnetically to obtain uniform irradiation of the whole sample con-
tents. The time-dependent fluorescence of DMA was measured while
the sample was illuminated by the laser. The fluorescence was ex-
cited at a wavelength in the range 375–400 nm, which was chosen
so that the optical density of DMA at this wavelength was lower
than 0.05. This assured a linear relationship between the concentra-
tion of DMA and its fluorescence intensity. Thus, the time-depen-
dent DMA fluorescence decrease reflected its time-dependent pho-
tosensitized destruction. The fluorescence of DMA was monitored
at wavelengths near 425–455 nm. There was no stray light leakage
from either the excitation light or from the illuminating laser. The
fluorescence traces were fitted to analytical functions by least
squares fitting programs.
The light-stability of the photosensitizers were evaluated in du-
plicate samples and under the same irradiation procedures as for
DMA photosensitizations. We measured the absorbance spectra of
H2F48Pc and ZnF48Pc at 736 and 705 nm, respectively, every 30 min
for 4 h under laser illumination. No decrease at the absorption bands
occurred.
Spectral resolution. The spectra of the partially or fully proton-
ated and deprotonated H2F48Pc along its titration process were ex-
tracted using principal component analysis (PCA) and singular value
decomposition (SVD) (3,4). A set of absorption, or fluorescence,
spectra are taken at increasing concentrations of added acid or base.
Each of the spectra is a linear combination of n ‘‘pure’’ spectra with
different weights (concentrations) Wn. If the number of contributing
species, n, is less than the number of measurements N, SVD of the
matrix containing the N measurements yields the eigenvalues (pure
spectra) of the n species. The theoretical concentrations of the spe-
cies are calculated from rate equations using the MATLAB function
roots (for finding all roots of a polynomial) and are fitted to the
12.3 g (95%) of white needles (m.p. 135.5–136.5ЊC). Anal. Found:
C, 36.82; H, 1.59; N, 5.27; Calc. for C16H8N2O4F12: C, 36.94; H,
1
1.55; N, 5.38%. H NMR (CDCl3
trimethylsilane [TMS], 400 MHz):
4.66 (q, 4H, JHF
8.06 Hz). 19F NMR (CDCl3
400 MHz): 87.02 (t, 6F, JHF 7.33 Hz), 87.42 (t, 6F, JHF
Hz). IR (KBr, cmϪ1): 2900 (
C–H), 2240 ( CN).
Synthesis of H2F48Pc. 3,4,5,6-Tetrakis-(2Ј,2Ј,2Ј-trifluoroe-
ϩ
␦
dimethylsulfoxide [DMSO]-d6,
4.50 (q, 4H, JHF 8.06 Hz),
DMSO-d6, C6F6,
7.33
ϭ
ϭ
ϩ
␦
ϭ
ϭ
␯
␯
thoxy)phthalonitrile (1.00 g, 1.92 mmol), lithium 18.7 mg (2.69
mmol), PhC(CF3)2OH 0.91 mL (5.4 mmol) and 1,8-diazabicy-
clo[5,4,0]undec-7-ene 0.21 mL (1.35 mmol) were added into anisole
(2 mL), and the mixture was reacted at 145–150ЊC for 60 h. After
cooling to room temperature ether (2 mL) was added to dissolve the
precipitated product, and finally the reaction was stopped by adding
2 N hydrochloric acid. The organic layer was extracted with ether
several times and the ether was evaporated. The resultant residue
was chromatographed using silica gel (Wako gel C-300) and meth-
ylene chloride–hexane (4:1 vol/vol), methylene chloride and finally
acetone–hexane (1:4 vol/vol). After removing the solvent by a rotary
evaporator the green residue was recrystallized from acetone–hexane
to give 0.628 g (63%) of the desired metal-free phthalocyanine.
Anal. Found: C, 37.20; H, 1.65; N, 5.27. Calc. for C64H34N8016F48:
1
C, 36.90; H, 1.66; N, 5.38%. H NMR (CD3CN, TMS):
␦
4.10 (q,
8.79 Hz). 19F NMR
8.55 Hz), 89.30 (t, 24F, JHF
NH), 2975 ( CH).
Synthesis of ZnF48Pc. In the above preparation of H2F48Pc, ZnCl2
95.4 mg (0.7 mmol) was used in place of lithium. After evaporation
16H, JHF
(CD3CN, C6F6):
8.79 Hz). IR (KBr, cmϪ1): 3300 (
ϭ
8.55 Hz), 5.54 (q, 16H, JHF
ϭ
␦
88.92 (t, 24F, JHF
ϭ
ϭ
␯
␯

Photochemistry and Photobiology, 2001, 73(5) 475
Table 1. Locations of absorption and fluorescence peaks of H2F48Pc and ZnF48Pc
H2F48Pc
ZnF48Pc
Deprotonated, Protonated,
Solvent
Acetone
Acetonitrile
Benzene
Chloroform
n-Decanol
Dioxan
Dimethylformamide
Ethanol
Ethylacetate
Hexane
n-Hexanol
Methanol
Nitrobenzene
Pyridine
Tetrahydrofuran
ET(30)
Absorption
Fluorescence
absorption
absorption
Absorption Fluorescence
42.2
45.6
34.3
39.1
47.7
36.0
43.8
51.9
38.1
31.0
48.8
55.4
41.2
40.5
37.4
702.8, 728.0
705.6
701.7
743.0
742.9
743.0
742.2
709.0
737.0
694.9
696.0
—
702.3
701.4
710.8
708.3
707.0
707.3
704.8
702.6
715.6
715.7
722.7
719.2
715.8
718.8
719.5
714.7
708.9, 736.8
704.1, 736.4
704.4, 733.4
708.0, 733.0
709.0, 731.2
699.0, 726.1
702.5, 731.0
701.5, 734.1
701.8, 731.0
698.3, 726.0
709.0, 736.5
792.0
702.0
699.4
687.2
699.0
692.9
693.4
701.5
699.7
687.2
700.8
699.0
696.2
—
—
—
793.8
739.7
740.0
739.2
705.8
705.7
701.6
716.6
716.6
720.9
—
797.0
708.0
740.9
707.9
703.7
718.7
716.1
705.0, 730.7
—
Ϫ
1
Ϫ1
extracted amplitudes using nonlinear least squares optimization. All
nonlinear least squares optimizations were done with lsqnonlin, a
strong (⑀ ഡ 24 000 M cm ), long wavelength bands as
classical QX(0, 0) and QY(0, 0) bands. The magnitude of the
split between these two bands in the various solvents is ap-
function from the MATLAB optimization toolbox. This function
2
finds the minimum of
⌺
n fn (p) where p is the set of fitted parameters
Ϫ
1
proximately 550 cm , which is much less than the usual
3000 cm 1 separation between these bands in porphyrins (5),
and the value of fn in our case is the difference between experimental
and theoretical value at the nth point. This function uses the inter-
ioreflective Newton method, with safeguarded mixed quadratic and
cubic polynomial interpolation and extrapolation method for a line
search algorithm.
Ϫ
but is similar to that obtained in other phthalocyanines (6).
This splitting is characteristic of the free base symmetry,
which is D2h and which thus eliminates the degeneration of
the Q transition into the two orthogonal X and Y transitions.
The two bands at shorter wavelengths are, again, vibrational
subband progressions, QX(0, 1) and QY(0, 1). The separation
between a main band and its progression, for H2F48Pc, is
RESULTS AND DISCUSSION
Absorption and fluorescence spectra
H2F48Pc and ZnF48Pc are sufficiently soluble in polar and
nonpolar organic solvents, as well as in other solvents, as
listed in Table 1. This better solubility than that of some
other metal-free and complexed phthalocyanines can be at-
tributed to the polarity of the fluoroethyl groups.
Ϫ
1
ϳ
1270
Ϯ
40 cm for [QX(0, 1)
Ϫ
QX(0, 0)] and
ϳ1460 Ϯ
Ϫ
1
40 cm for [QY(0, 1)
Ϫ QY(0, 0)]. The splitting is absent
in ZnF48Pc, which possesses D4h symmetry and therefore has
full in-plane symmetry which degenerates the X and Y tran-
sitions (5,7). The major emission band of H2F48Pc is located
in benzene (Fig. 2) at 743 nm and that of ZnF48Pc is at 723
nm. As in other porphyrins and phthalocyanines the peak-
to-peak Stokes shift between the absorption and emission
Figure 2 shows the absorption and fluorescence spectra of
H2F48Pc and ZnF48Pc in benzene. A few features in these
spectra are worth mentioning. ZnF48Pc is seen to have a very
intense and narrow long-wavelength Q band, located at 711
Ϫ
1
Ϫ
1
Ϫ1
bands is small, near 100 cm for the metal-free compound
nm, and an ⑀ Ͼ 65 000 M cm . The smaller band of
ZnF48Pc, which is seen at shorter wavelengths (at 640 nm in
benzene), is a vibrational progression of the Q band, sepa-
Ϫ
1
and 250 cm for the zinc complex. Unlike metal-free and
metal-bound tetrabenzoporphyrin (8), the Soret bands of the
fluorinated compounds are weak, very broad and ill defined.
Table 1 summarizes the location of the main absorption
and fluorescence peaks of H2F48Pc and ZnF48Pc in various
solvents. The location of the Q bands of H2F48Pc and
ZnF48Pc are seen to change between the solvents. This prop-
erty of solvatochromism, caused by differential solvation of
the ground and first excited state of the light absorbing mol-
ecule, is observed for many compounds (9–13). We used
two common approaches to model and evaluate the effect of
the solvents on the absorption spectra: (1) the relation be-
tween the peak wavelength of the longest wavelength Q
band and the ET(30) scale of Dimroth and Reichardt. This
parameter is based on the polarity of the solvents, as reflect-
ed in the absorption spectra of a single probe, commonly
denoted as Reichardt’s dye (12); and (2) the three-parameter
approach of Kamlet, Abboud and Taft (KAT). This method
Ϫ
1
rated from the main band by 1550 cm . In contrast, the free
H2F48Pc exhibits a system of four split Q bands, appearing
in benzene at 644, 674, 709 and 737 nm. We assign the two
Figure 2. Absorption and fluorescence spectra of H2F48Pc and
ZnF48Pc in benzene. The concentrations were 3
␮M. The fluores-
cence spectra were excited at 709 nm (H2F48Pc) and at 640 nm
(ZnF48Pc) and are normalized to equal height.
assigns each solvent three characteristic parameters: ␲*, an

476 Hana Weitman et al.
Table 2. The best fitted equations for the absorption spectra of H2F48Pc and ZnF48Pc and the standard deviation of the fits
Model
Compound
ET(30)
0.2374·ET(30)
KAT
ZnF48Pc
H2F48Pc
␭
ϭ
715.4
0.00282
747.28
0.00308
713.64
0.00488
Ϫ
␭
ϭ
707.28
0.00307
735.01
0.00297
699.02
0.00435
ϩ
ϩ
Ϫ
0.0788
0.3671
3.4082
␲
␲
␲
*
*
*
Ϫ
Ϫ
Ϫ
2.1092␣ Ϫ 3.4374
2.8742␣ Ϫ 7.3045
5.6329␣ ϩ 0.4036
␤
␤
␤
max
max
␭
max
ϭ
Ϫ
0.3739·ET(30)
0.3873·ET(30)
␭
max
ϭ
Ϫ
F48Pc2
␭
max
ϭ
Ϫ
␭
max
ϭ
index of solvent dipolarity/polarizability, which relates to the
ability of the solvent to stabilize a charge or a dipole by
judged by the error, is similar to that of the single-parameter
method based on the ET(30) scale, which is based only on
the polarity of the solvents (12).
virtue of its dielectric effect;
␣
, a parameter which reflects
, which
the solvent’s hydrogen-bond donor acidity; and
␤
scales the solvent’s hydrogen-bond acceptor basicity, pro-
viding a measure of the solvent’s ability to solubilize the
solute by accepting a proton through a hydrogen bond
(14,15).
Protonation and deprotonation in organic solvents
Metal-free phthalocyanines contain four pyrrole nitrogen at-
oms, all of which are theoretically capable of undergoing
protonation/deprotonation reactions. The acid–base proper-
ties of porphyrins were studied and reviewed extensively in
terms of the pK of these nitrogens and the effect of the
protonation state on the absorption spectrum and on the up-
take by liposomes and cells (17–20). The last issue is of
great relevance for the application of porphyrins and phtha-
locyanines as biological photosensitizers, especially in the
photodynamic therapy of cancer. The protonation of the pyr-
role nitrogens, as well as of various side groups, strongly
affects the uptake of the molecules by the external mem-
brane of the cell as well as the transport through the mem-
brane (21–25). This leads to a pH effect on the photosensi-
tizing capacity in cells and in tissue (26,27). Chloraluminum
phthalocyanine was observed to exhibit an increase in its
cellular photokilling effect upon decreasing the pH from 8
to 6. This effect was attributed to either an intrinsic effect
of protonation on photosensitization efficiency or due to pH-
dependent interaction with cellular sites (28).
The protonation state of the pyrrole imine groups affects
the absorption spectrum, namely the number, location and
strength of the Q bands. Metal-free porphyrins at neutral pH
in aqueous solutions, whose pyrrole core is electroneutral
with two protonated nitrogens, exhibit four Q bands in the
500–630 nm range, due to the symmetry and due to the
vibrational splitting, as described above. Complete proton-
ation or deprotonation of the four pyrrole nitrogens restores
the D4h symmetry and two Q bands are observed (18,29–
31). This spectral characteristic is also the proof for the pyr-
role nitrogens being protonated (32) and not the outer, bridg-
ing nitrogens (33).
␭ ϭ ␭ ϩ
s␲*
ϩ
a␣ ϩ
b
␤
(1)
0
We performed least squares fitting of the spectral data to
ET(30), while for the multiparameter KAT method, we car-
ried out multiparameter linear regression of the correlation
between the peak of the longest wavelength Q band and the
tabulated parameters ␣, ␤ and ␲* (15,16). Table 2 gives the
fitted parameters of the two solvatochromism models and the
error. The error in this table is the average deviation of the
fitted wavelength from the measured one, relative to the
measured wavelength. As can be observed in Table 1 and
from the solvatochromic equations in Table 2, the solvato-
chromic effect is not large. The bands of H2F48Pc and
ZnF48Pc have a maximal change of
ϳ10 nm between their
extreme values. This indicates that the molecular electronic
charge distributions in the ground and excited electronic
states are not considerably different. This can be rationalized
especially with the more symmetrical ZnF48Pc. The negative
solvatochromism that we observe in all cases, namely a hyp-
sochromic shift, to shorter wavelengths, upon increasing the
solvent’s polarity, shows that the ground state is more di-
polar than the excited state. Surprisingly, it is seen that even
though the KAT model diversifies its dependence on three
independent parameters, the quality of the fit to the data,
Spectral evidence of the titration of porphyrins in organic
environments is very rare, and is even rarer for phthalocy-
anines. The perfluorination in the studied molecule could
render the pyrrole nitrogens more acidic by stabilizing the
unprotonated anion. Due to the very low solubility in water
we could not carry out a titration reaction in water and obtain
a pK value. Rather, we measured the spectra of H2F48Pc in
the organic solvents, upon addition of TFA or TEA. As can
be observed in Table 1 and Fig. 3 the addition of the organic
base TEA caused the absorption spectrum to possess only
Figure 3. Absorption spectra of H2F48Pc (3
(- - -) and with excess TFA (—) and excess of TEA (– – –).
␮M) in nitrobenzene

Photochemistry and Photobiology, 2001, 73(5) 477
two Q bands, with the strong one shifted to shorter wave-
lengths by 40 nm, relative to the QX(0, 0) band. The pro-
tonation of H2F48Pc by the addition of the acid TFA caused
ϳ
a bathochromic shift of
one Q band.
ϳ50 nm, with the appearance of only
In the case of the four solvents acetone, acetonitrile, DMF
and pyridine, we observed a peculiar behavior. Native
H2F48Pc in these solvents gave only two Q bands, with the
strong one located in the range where the band of the TEA-
deprotonated species appears in the other solvents. This in-
dicates that in these four solvents the molecule is deproton-
ated. This is corroborated by the fact that the addition of
TFA to acetone and DMF generates four Q bands located at
wavelengths that would be expected from H2F48Pc.
Of the various solvents that we used, only nitrobenzene
allowed full titration of H2F48Pc in both directions, namely
with TFA or with TEA. This arises probably from the strong
polarity of this aprotic solvent. Figure 3 shows the absorp-
tion spectrum of free H2F48Pc in nitrobenzene and when TFA
and TEA were added. The spectral changes are fully revers-
ible.
Figure 4. (A) Absorption spectra of native H2F48Pc, H3F48Pcϩ and
ϩ
H4F48Pc2 as obtained from SVP of absorption measurements in the
titration of H2F48Pc with TFA in nitrobenzene. (B) The calculated
fractions of each of the species as a function of TFA concentration.
librium constants. Nonlinear regression was employed to
calculate the equilibrium concentrations of the species of P
by minimizing the difference between the measured spectra
along the titration and the calculated sum of species contri-
butions to these spectra. SVD decomposition of 20 measured
absorption spectra upon acid titration revealed that there
were three significant components, shown in Fig. 4a. The
first is native H2F48Pc. The second resolved component aris-
ϩ
An issue that has been discussed in regard to the acidity/
basicity of tetrapyrroles is the question whether the mono-
es from the monoprotonated species H3F48Pc and is seen to
still have two major Q bands and two progressions, since
the D4h symmetry is still not existent. The third component,
which appeared at higher TFA concentrations, was
ϩ
Ϫ
cation (H3F48Pc ) or monoanion (H1F48Pc ) can be observed
in the acid or base titration, respectively. It is possible that
the second step in the titration is energetically more favor-
able than the first, leading immediately to the dication
ϩ
H4F48Pc2 , with characteristic D2h symmetry features. Once
the three contributing pure spectra are known we calculated
their contributing weights in each one of the full set of 20
measured spectra. Figure 4b shows the relative weight (con-
centration) of the three species as a function of acid concen-
tration. It is clearly observed that the monoprotonated mol-
ecule is generated upon acidification and later its concentra-
tion diminishes, causing the diprotonated species to exhibit
a lag in its build-up.
ϩ
Ϫ
(H4F48Pc2 ) or dianion (F48Pc2 ). Almost all studies were
carried out in aqueous solutions or in protic solvents, and
contradicting results were obtained regarding the presence
of the monocation or monoanion (17–19,30,31,34–36). Mon-
ocations of porphyrins have also been observed to exist in
aqueous solutions of detergents (37).
We thus carried out a stepped titration of H2F48Pc in ni-
trobenzene with TFA or TEA, to investigate the molecule’s
basicity or acidity. The large set of absorption spectra was
analyzed by SVD. This is done to obtain the number and
shape of the basic spectra, which comprise, by their linear
combinations, all the measured spectra, and to determine
also the acid–base equilibrium constants described below.
We thus followed the procedure described in the following
paragraphs.
Acid titration with TFA. To evaluate the titration of
H2F48Pc (P) with acid (A) and its base conjugate (B), we
considered the following reactions and their equilibrium con-
stants:
The concentration of each of the basic components can be
used to evaluate the equilibrium constants. However, the in-
ϩ
creasing concentration of the diprotonated H4F48Pc2 is sta-
tistically more reliable than the concentrations of the original
ϩ
H2F48Pc and the monoprotonated H3F48Pc because its spec-
trum is very different from the spectra of the other two. We
Ϫ
10
Ϫ4
thus found that KH1
ϭ 2.7 ϫ 10 and KH2 ϭ 3.2 ϫ 10 .
These constants can be affected by moisture present in ni-
trobenzene, even of spectral grade. However, since all mea-
surements were carried out in nitrobenzene of the same
batch, a comparison of the constants is justified. These con-
stants indicate that the second stage of protonation over-
whelms the first one. Therefore the equilibrium concentra-
k01
k01
ϩ
tion of the doubly protonated H4F48Pc2 is higher, under
ϩ
Ϫ
P
ϩ
ϩ
A
s
P
ϩ
B
ϭ
ϭ
KH1
KH2
ϩ
k10
k10
most conditions, than that of the monoprotonated H3F48Pc .
Basic titration with TEA. As in the previous discussion
we considered similar reactions and their equilibrium con-
stants of the deprotonation of H2F48Pc (P) by a base to form
k12
k12
k21
ϩ
ϩ
Ϫ
P
A
s
P2
ϩ
B
k21
Ϫ
Ϫ
P and P2 . We measured 14 absorption spectra during the
titration at different concentrations of TEA. SVD, which was
carried out on the whole set of spectra or on those obtained
at either small or large concentrations of TEA, revealed only
two significant components. One is native H2F48Pc and the
second one looked very similar to the last-measured spec-
trum in the series, where a large excess of base was present.
The resolved spectra are shown in Fig. 5a. The nonlinear
least squares analysis also gave the weights by which these
kt
ϩ
ϩ
P
ϩ
ϩ
P2
s
2P
kϪ
t
kb
ϩ
Ϫ
P
2A s ϩ 2B
P2
kϪ
b
We then write the equations for the equilibrium concentra-
ϩ
ϩ
tions of P, P and P2 as a function of the total concentra-
tions of the phthalocyanine and the added acid and the equi-

478 Hana Weitman et al.
kDMA
΂ ΃
kpho
sens
⌽
⌽
⌬
⌬
,sens
,stan
ϭ
(3)
(4)
kDMA
΂ ΃
kpho
stand
Ϫ
10(
Ϫ
abs·L)
0.97·P·(1
kpho
ϭ
E·V
Figure 5. (A) Absorption spectra of native H2F48Pc, HF48PcϪ and
Ϫ
F48Pc2 as obtained from SVP of absorption measurements in the
P is the laser’s power (in mW), abs is the optical density of
the reaction sample at the wavelength of irradiation, L is the
sample’s pathlength through which the laser beam travels, E
titration of H2F48Pc with TEA in nitrobenzene. (B) The calculated
fractions of each of the species as a function of TEA concentration.
is einstein units (1 einstein
ϭ 6.023 ϫ
1023 photons) of light
energy per second per watt of light at the irradiating wave-
length and V is the volume of the sample solution in milli-
liters. The factor 0.97 corrects for the light reflected at the
air/sample interface (38).
We measured the singlet oxygen production yield in ben-
zene and liposomes. TPP was used as a standard in benzene,
two spectra compose the five analyzed spectra. Figure 5b
shows the concentration of the two species as a function of
TEA concentration. The diminishing concentration of
H2F48Pc and the increase of the rising species are fitted well
Ϫ
if this species is assumed to be the dianion, F48Pc2 . This fit
also yields the concentration and spectral shape of the mon-
oanion which cannot be resolved directly by SVD.
where its
liposomes, with ⌽⌬ ϭ 0.75. In benzene
and 0.58 0.01 for H2F48Pc and ZnF48Pc, respectively. In
liposomes, was 0.005 0.0001 and 0.35 0.01. When
⌽ is 0.64 (39) and HP served as a standard in
⌬
⌽
⌬
was 0.16 Ϯ 0.01
The equilibrium constant for the first deprotonation is KB1
Ϯ
Ϫ
ϭ
is KB2
1.8
ϫ
ϭ
10 10 and the constant for the second deprotonation
⌽
Ϯ
Ϯ
⌬
Ϫ
3
5.3
ϫ 10 . The ratio of the second deprotonation
singlet oxygen is generated in a liposome it can diffuse rap-
idly into the aqueous medium (40) and escape the mem-
equilibrium constant, relative to the first, is about 25 times
larger than the respective ratio in the protonation process.
This can be explained on the basis of the electron withdraw-
ing power, exerted by the fluorination, which stabilizes and
delocalizes the negative charges that are created upon de-
protonation.
brane-bound DMA trap. With a diffusion constant of 1.5
ϫ
Ϫ
5
Ϫ1
10 cm2 s (in DMPC liposomes at 21
Њ
C), oxygen can
diffuse 50 nm within 1
ϳ
␮s. Kanofsky (41) estimated the
effective lifetime of singlet oxygen in red blood cell ghosts
to be 24–130 ns. Indications for rapid diffusion of singlet
oxygen out of the membranes of liposomes and cells was
demonstrated by Kanofsky and coworkers (42–44). Thus, a
measurement of singlet oxygen yield in membranes, which
is done by employing chemical targets, represents an effec-
tive yield in terms of causing chemical damage to the spe-
cific target which is employed, relative to the specific stan-
dard which is employed. We see that in benzene, as well as
in liposomes, ZnF48Pc has a much higher yield than H2F48Pc,
reflecting the metal ion’s effect on intersystem crossing into
the excited triplet level.
Photosensitization efficiency
The fluorinated phthalocyanine possesses a feature that is
important for its possible application to photosensitization,
namely, a strong absorption band at long wavelength. This
makes this molecule appealing for biological photosensiti-
zation, because of deeper penetration of longer wavelengths
into scattering media. For the determination of the singlet
oxygen quantum yield, ⌽⌬, we used DMA as a chemical trap
for singlet oxygen. DMA is a very suitable target because it
reacts very efficiently and selectively with singlet oxygen;
its concentration can be followed conveniently by its fluo-
rescence, which disappears upon peroxidation; and its fluo-
rescence is not affected by the sensitizer. The weakness of
the fluorescence of DMA in water is very important when
studying sensitization in a membrane, because the measured
fluorescence reflects mainly membrane-localized DMA. The
decrease in the fluorescence of DMA during photosensiti-
zation by the phthalocyanines was always found to be ex-
ponential, from which the first-order decay kinetics constant
was calculated by fitting to:
We also wanted to compare the photosensitizing efficien-
ϩ
cies of native H2F48Pc and the protonated H4F48Pc2 and un-
Ϫ
protonated F48Pc2 . The only solvent in which all three spe-
cies can be obtained is nitrobenzene (see spectra in Fig. 3).
As a chemical target for singlet oxygen we chose rubrene
which exhibits a large reaction constant in many solvents.
DMA could not be used because its absorption and fluores-
cence are masked by the absorption of nitrobenzene. We
monitored rubrene’s photosensitized destruction by follow-
ing its absorption bands, at 497 and 535 nm, which are not
masked by the edges of the absorption of nitrobenzene itself.
We could not find any sensitizer whose singlet oxygen pro-
duction yield in nitrobenzene is known, thus the following
ϪkDMA·t
FDMA
ϭ
F0 ·e
(2)
results are relative values:
⌽
(H2F48Pc)
ϭ
1.000;
(H4F48Pc2 )
ϭ 0.028; ⌽ Ͻ 0.001. Surprisingly,
⌬
⌽
⌬
⌬
Ϫ
ϩ
(F48Pc2 )
where kDMA is the rate constant for the disappearance of
protonation or deprotonation strongly diminishes singlet ox-
DMA’s fluorescence, FDMA. The singlet oxygen quantum
Ϫ
ygen’s production. The low yield by F48Pc2 was indepen-
yield,
⌽⌬, of the sensitizer is calculated relative to that of a
dent of its concentration. This rules out the possibility of
intermolecular quenching, which has been demonstrated pre-
viously with anions such as superoxide, azide and iodide
known standard, in the same solvent, after proper correction
for the extent of absorption of the laser’s photons by the
sensitizer and the standard, kpho, using the following expres-
sion:
(45,46). The changes in
⌽ must thus be due to an intrinsic
⌬

Photochemistry and Photobiology, 2001, 73(5) 479
Table 3. NMR chemical shifts for H2F48Pc*
13C (185 K)
13C (330 K)
1H (300 K)
19F (297 K)
1
2
3†
4†
CH2
157.59, 141.14
127.81, 122.55
148.76, 148.10
146.72, 146.55
72.70, 72.56
149.52
125.52
148.39
146.73
73.21, 71.76‡
5.05, 5.58§
71.14, 71.00‡
125.28, 125.02
CF3
NH
࿣
125.08, 124.67
࿣
Ϫ72.53, Ϫ72.97§
Ϫ0.05
*In THF-d8, see ‘‘Materials and Methods.’’
†Assignments may be interchanged.
‡2JCF
1JCF
§3JHF
ϭ
ϭ
ϭ
35 Hz.
279 Hz.
8.5 Hz.
࿣
property of the sensitizer in its various states of pyrrole pro-
tonation.
In reality, therefore, there are only two types of phthalate
moieties, B/D and A/C, which differ in that the nitrogen
atom is or is not protonated, respectively; both types are
symmetrical, i.e. one expects to see only four types of ring
carbons for each.
The existence of a tautomerization process that intercon-
verts the two types of units in porphyrin-like systems by
exchanging the pair of hydrogens between the four central
nitrogen atoms has been known for many years (47–49). The
thermodynamic parameters for this process have been in-
vestigated several times by NMR lineshape analysis tech-
niques ([50–54] and references therein).
NMR spectroscopy
A cursory observation of the structure of a phthalocyanine
such as H2F48Pc (see below) might lead to the conclusion
that there are three different types of phthalate moieties: A/
C, B and D, with the first type being asymmetrical (eight
different types of ring carbons). Actually, the
␲ system is a
resonance hybrid, and the structure as drawn, with a specific
positioning of the double bonds, is only one of the possible
canonical structures (Scheme 1).
The NMR spectra (the data are summarized in Table 3)
of H2F48Pc at room temperature indicate that this tautomer-
ization process is in fact quite fast in the NMR time scale;
1
it is manifested only in a slight broadening of the H and
19F lines. In the room temperature 13C spectrum, however,
one of the ring carbon signals is too broad to escape obser-
vation (this phenomenon was described also by Cook et al.
[55] for an octaalkylphthalocyanine). Only by heating the
solution a broad line appears at
␦ 149.52. At low tempera-
tures most carbon peaks split into two lines of equal intensity
(see Table 3). In the intermediate temperature regime it is
possible to fit the experimental NMR lineshapes to calculat-
ed traces (obtained by simulating the lineshapes of coalesc-
ing pairs of carbon lines with a computer program based on
the equations described by Sutherland) (56). Thus, the rate
constants for this process could be deduced (the results are
presented in Table 4; the pairs of lines with a very large
chemical shift difference, corresponding to carbons 1 and 2,
see Table 3, were useful for the determination of the rate
constants at the higher temperatures).
Table 4. Dynamic NMR-derived thermodynamic parameters for
the tautomerization process of H2F48Pc*
As far as we could ascertain, no such measurement has
been performed for a phthalocyanine in solution. A paper by
Wehrle and Limbach (57) described a similar study for 15N-
enriched phthalocyanine in the crystalline state in which rate
constants are determined for each of two crystalline forms
1
T (K)
k (secϪ
)
⌬
G† (kcal molϪ1)†
216.5
232.3
247.1
271.8
299.2
330.2
90
500
2500
10.60
10.60
10.53
10.89
11.15
11.42
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
Ϯ
0.12
0.12
0.20
0.20
0.17
0.27
10 000
45 000
190 000
Ϫ
1
(with ⌬
G† values in the 9–10 kcal mol range).
Acknowledgements We wish to acknowledge the kind and gener-
ous support (Grant 9800364) of the United States—Israel Binational
Science Foundation (BSF), Jerusalem, Israel. We are glad to ac-
knowledge the support by the David Falk Chair in Laser Photother-
apy (B.E.).
1
*In THF-d8.
⌬
.
H†
ϭ
9.0
Ϯ
0.5 kcal molϪ
,
⌬S† ϭ Ϫ7.2
Ϯ
2.0 kcal
1
1
molϪ KϪ
†⌬G† values were calculated with the Eyring equation.

480 Hana Weitman et al.
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