A laser flash photolysis study of fenofibric acid in aqueous buffered media: Unexpected triplet state inversion in a derivative of 4-alkoxybenzophenone

Article abstract of DOI:10.1562/0031-8655(2002)075<0193:ALFPSO>2.0.CO;2

Laser excitation of aqueous solutions of fenofibric acid (FA) at pH 7.4 show the formation of two reaction intermediates, the triplet state and the hydrated electron. The former is longer lived in water than in acetonitrile; its anionic form decays irreve

Full text of DOI:10.1562/0031-8655(2002)075<0193:ALFPSO>2.0.CO;2

A Laser Flash Photolysis Study of Fenofibric Acid in Aqueous Buffered Media:
Unexpected Triplet State Inversion in a Derivative of 4-Alkoxybenzophenone
Author(s): Gonzalo Cosa, S. Purohit, J. C. Scaiano, Francisco Boscá, and Miguel A. Miranda
Source: Photochemistry and Photobiology, 75(3):193-200.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2002)075<0193:ALFPSO>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282002%29075%3C0193%3AALFPSO
%3E2.0.CO%3B2
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Photochemistry and Photobiology, 2002, 75(3): 193–200
A Laser Flash Photolysis Study of Fenofibric Acid in Aqueous Buffered
Media: Unexpected Triplet State Inversion in a Derivative of
4-Alkoxybenzophenone¶
Gonzalo Cosa1,2, S. Purohit1, J. C. Scaiano*1, Francisco Bosca´2 and Miguel A. Miranda2
1Department of Chemistry, University of Ottawa, Ottawa, Canada and
2Departamento de Qu´ımica/Instituto de Tecnolog´ıa Qu´ımica UPV-CSIC, Universidad Polite´cnica de Valencia, Valencia,
Spain
Received 2 October 2001; accepted 7 December 2001
ABSTRACT
combined effects of UVA and FA can result in photosensi-
tized DNA damage (5), peroxidation of fatty acids (6) and
red blood cell hemolysis (1). However, its phototoxic effect
is reportedly lower compared with another benzophenone-
derived drug, ketoprofen (5). The reactivity of FA and FB
in ethanol suggest an n,␲* lowest triplet state, in agreement
with calculations (7). No such reactivity has been measured
in water.
Laser excitation of aqueous solutions of fenofibric acid
(FA) at pH 7.4 show the formation of two reaction inter-
mediates, the triplet state and the hydrated electron. The
former is longer lived in water than in acetonitrile; its
anionic form decays irreversibly by decarboxylation to
give a carbanion that protonates before or after rear-
rangement. Several spectroscopic and quenching studies
suggest that in aqueous media the triplet state of FA has
a
␲,␲* character, in comparison with an n,␲* character
in organic media. Further, the known chemistry of the
triplet, including decarboxylation and hydrogen abstrac-
tion, occurs predominantly from the n,
consequence, activation energies are higher when the
lowest triplet has a * character. Photoionization is
␲* state, and as a
␲,␲
more important in aqueous than in organic media and
involves a biphotonic process. Hydrated electrons are
trapped by FA, leading to the corresponding ketyl radical
after protonation.
In this work we present laser flash photolysis (LFP) stud-
ies performed in aqueous buffered solutions and compare
with acetonitrile:water mixtures. We analyze the reactivity
toward decarboxylation and hydrogen abstraction of FA and
its ester form, FB. We also report triplet-energy values as
well as spectroscopic characteristics of the T-T absorption
band for FA and FB, measured over a wide solvent polarity
range.
INTRODUCTION
The photochemistry of pharmaceutical products has received
much attention in the last decade, prompted by reports on
photosensitization reactions produced by certain active in-
gredients in drugs. Those related to the benzophenone chro-
mophore have been studied in great detail, and a recent re-
view has been published on the topic (1).
Fenofibric acid (FA) is a hypolipidemic drug circulating
in the blood stream after the metabolism of the commercial
prodrug fenofibrate (FB) (2,3). This molecule consists of a
substituted benzophenone, its ground state absorption ex-
tending over the UVA part of the solar spectrum (4). The
Our studies are aimed at understanding the photophysical
and photochemical behavior of FA in water. All the param-
eters measured indicate that an inversion of the lower triplet
state of fenofibric acid (n,␲* to ␲,␲*) occurs when going
from acetonitrile to water; this would account for the lower
reactivity of FA and its photoproducts as compared with
those of ketoprofen in aqueous media.
Our results also show that biphotonic processes occur un-
der pulsed laser excitation, where solvated electrons, togeth-
er with ketyl and cyclohexadienyl radicals, are readily ob-
servable.
¶Posted on the website on December 31, 2001.
*To whom correspondence should be addressed at: Department of
Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, ONT
K1N 6N5, Canada. Fax: 613-562-5633; e-mail: tito@photo.
chem.uottawa.ca
Abbreviations: FA, fenofibric acid; FB, fenofibrate; LFP, laser flash
photolysis; 1-NM, 1-naphthalenemethanol; TMAOH, tetrameth-
ylammonium hydroxide.
MATERIALS AND METHODS
Materials. FB (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic
acid isopropyl ester), tetramethylammonium hydroxide (TMAOH),
sodium phosphate (monobasic), sodium hydroxide (semiconductor
grade), sorbic acid (2,4-hexadienoic acid), 1-naphthalenemethanol
(1-NM) and ethanol were purchased from Sigma-Aldrich Canada
᭧
2002 American Society for Photobiology 0031-8655/02
$5.00ϩ0.00
193

194 Gonzalo Cosa et al.
5
5
ϫ
10Ϫ M FA in
Figure 1. Transient absorption spectrum of 3.5
ϫ
10Ϫ M FA in
Figure 2. Transient absorption spectrum of 3.5
0.01 M phosphate buffer solution of pH 7.4; laser at 308 nm; N2
0.01 M phosphate buffer solution of pH 7.4; laser at 308 nm; N2
bubbled. Time windows: ( ) 0.04 s; ( ) 0.40 s; ( ) 1.28 s;
s after the laser pulse. Inset: kinetic profile at 340
᭢
␮
᭝
␮
ⅷ
␮
bubbled. Time windows: (
after the laser pulse. Also shown is an enlarged section of the 8
time window ( ).
⅜) 8
␮s; (Ⅺ
) 139
␮s and (
ࡗ
) 1740
␮
␮
s
s
and (Ⅺ) 4.04 ␮
nm.
⌬
(Oakville, Canada). All solvents were high-performance liquid chro-
matography (HPLC)–grade and purchased from OmniSolv (Gibss-
town, NJ) and were used without further purification. Water was
purified through a Millipore MilliQ system. The 0.1 M, pH 7.4 phos-
phate buffer was prepared with monobasic sodium phosphate in wa-
ter, and the solution was brought to pH with NaOH.
Steady state measurements. Phosphorescence spectroscopy was
performed using a Perkin Elmer LST 50 luminescence spectrometer.
The spectra were recorded at 77 K in different glasses. Absorption
spectra were recorded using a Varian CARY 1E spectrophotometer.
Product studies. Photolysis of FA in phosphate buffer aqueous
solutions was done with 308 and 355 nm lasers as the excitation
sources. The samples received 100 laser shots and were then ex-
tracted with dichloromethane. The solvent was removed under vac-
uum, and the resulting mixture was analyzed with a Fisons GC/MS
FA (2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid)
was obtained after hydrolysis of the ester form, FB, with NaOH (2.7
M) in 50:50 H2O–methanol under reflux for 5 h. After methanol
evaporation, the aqueous mixture was subsequently extracted with
ether (three washes) to remove any nonreacted FB. The aqueous
phase was then brought to pH 5 with a 1:10 HCl–H2O mixture. Ethyl
acetate was then used for another series of extractions (three
washes); the organic phase containing the FA was dried with
NaHSO4 and filtered and the solvent evaporated under low pressure.
The product was a white powder that was confirmed to be FA by
8060/8000 Series equipped with a J&W DB5 30 m
column.
ϫ 0.32 mm
HPLC studies of the irradiated solutions were carried out on a
Varian instrument using a reverse phase Zorbax SB-C18 column;
the mobile phase used was 15:85:1 water:methanol:acetic acid. The
detection used a Varian 9065 Polychrom diode array detector. The
absorption spectra of the products were compared with those re-
ported previously (6).
1
comparing the H-NMR (CDCl3) spectra taken before and after the
saponification, showing the disappearance of the peaks correspond-
Quantum yield determination. Steady state photolysis was carried
out in a photoreactor, with only one 254 nm 8 W mercury lamp and
for the periods described in Fig. 10. Ketoprofen was employed as a
standard (the quantum yield of photodecarboxylation is 0.75 at pH
7.4) (10). The solutions were 1 mM in the substrate and 80 mM in
TMAOH, to ensure that all the photons would be absorbed under
these conditions and that FA and ketoprofen would be in their an-
ionic forms. To quantitate the starting material as well as the pho-
toproducts produced, a calibration curve was made with FA at 254
nm and employed equally for FA and for photoproduct 1, which has
the same chromophore structure. In the case of photoproduct 2, the
concentration was corrected for the difference in molar absorptivity
existing between FA and this compound at 254, as reported in the
literature (6). A separate curve was prepared for ketoprofen.
ing to the isopropyl group (␦ 1.2 [d, 6H, J1 ϭ 6 Hz], 5.1 [m, 1H]).
Nanosecond LFP. The LFP system has been previously described
(8,9). Samples were excited either with a Lumonics EX-530 laser
with a Xe–HCl–Ne mixture generating pulses of 6 ns at 308 nm
with
Յ100 mJ output at the source or with the third harmonic of a
Surelite Nd:YAG laser generating pulses of 8 ns duration at 355 nm
with 20 mJ output. The signals from the monochromator–photo-
multiplier system were initially captured by a Tektronix 2440 digi-
tizer and transferred to a PowerMacintosh computer that controlled
the experiment with software developed in the LabVIEW 4.1 envi-
ronment from National Instruments (Austin, TX).
All transient spectra and kinetics were recorded employing a flow
system with a 7
ϫ 7 mm, 2 mL capacity fused silica cuvette from
Luzchem Research (Morin Heights, Canada). Samples were purged
in a storage tank for 30 min either with N2, O2 or N2O. The quench-
ing rate constants were obtained with static samples contained in 7
RESULTS
ϫ
7 mm fused silica cuvettes. A fresh solution was employed to
obtain each decay rate; two to four laser shots were delivered to the
sample and the solution was shaken between shots to avoid any
interference caused by the accumulation of photoproducts. Each so-
lution was purged for 15 min with N2, O2 or N2O before data ac-
quisition.
FA concentration was ca. 0.035 mM for irradiation at 308 nm.
When the excitation was carried out at 355 nm, the sample concen-
tration was ca. 1 mM. These values ensured an absorbance of 0.3
to 0.5 in the laser cell at the excitation wavelength.
Aqueous solutions of FA were prepared in pH 7.4 buffers, 10 and
100 mM in phosphate, for irradiation at 308 and 355 nm, respec-
tively. The solutions prepared in methanol or acetonitrile:water had
10 mM TMAOH to ensure that FA was present in its dissociated
form.
In this work, we have examined the photochemistry of FA
in aqueous media. FA is the biologically active drug pro-
duced by hydrolysis of FB. Most of the work was carried
out at pH 7.4 to approximate physiological conditions.
Laser excitation of FA at pH 7.4 under nitrogen leads to
the spectrum of Fig. 1. The early spectrum corresponds to a
mixture of two species, as revealed by a number of confir-
matory experiments (vide infra). The bands at 360 and 460
nm are assigned to the triplet state of FA, which at this pH
is present as the anion, whereas the absorption at ␭ Ͼ 700
nm is probably dominated by the hydrated electron, with
contributions from triplet absorption. At 340 nm, we also
observe the formation of another species after triplet decay
(Figs. 1 and 2). The fact that the time profile for the change
The 355 nm pulses from a Nd:YAG laser were used to determine
quenching rate constants with 1-NM, in order to avoid absorption
by the quencher.

Photochemistry and Photobiology, 2002, 75(3) 195
4
Figure 4. Transient absorption spectra of 8.9
ϫ
10Ϫ M FA in 0.1
Figure 3. Electron absorption spectrum obtained from the difference
between the spectrum of 3.5
buffer of pH 7.4; laser 308 nm, N2 bubbled; and the spectrum of a
solution under the same conditions but equilibrated with N2O. The
M phosphate buffer solution of pH 7.4. The quencher used was 2
ϫ
5
ϫ
10Ϫ M FA in 0.01 M phosphate
3
10Ϫ M 1-NM; laser 355 nm; N2 bubbled. Time windows: (
ⅷ) 29
ns; (
Ⅺ
) 200 ns; (
ϩ
) 425 ns; and (
⌬
) 680 ns after the laser pulse.
Inset: kinetic traces obtained at (
ⅷ
) 480 nm and at (Ⅺ) 420 nm.
observed time windows are those registered 0.16
pulse. The spectra were normalized to the same value at 380 nm
before subtraction. ( ) N2O; ( ) N2; ( ) solvated electron. Inset:
transient absorption spectrum of 3.5
10Ϫ M FA in 0.01 M phos-
phate buffer solution of pH 7.4; laser 308 nm, N2O bubbled. Time
windows: ( ) 8 s and ( ) 344 s after the laser pulse.
␮s after the laser
ⅷ
Ⅺ
ϩ
these intermediates need to be transparent or at least less
absorbing than both the triplet state and the species respon-
sible for the signal growth. In our case, the simplest hypoth-
esis is that the 340 nm signal is derived from the hydrated
electron. This is confirmed by the fact that electron scav-
enging with N2O eliminates the growth at 340 nm. The 340
nm transient, which also shows a signal at 540 nm (see Fig.
2), is assigned to the ketyl radical formed by reduction of
ground state FA after trapping of the hydrated electron. Its
absorption agrees well with the spectrum of the acid form
5
ϫ
Ⅺ
␮
ࡗ
␮
in absorbance (⌬OD) shows a valley at 340 nm implies that
the triplet state cannot be the immediate precursor of this
species (see the inset of Fig. 1).
In order to verify the assignment of the long wavelength
signals to the hydrated electron, we carried out experiments
in N2O-saturated solutions. This is a common diagnostic test
for hydrated electrons, which are efficiently scavenged by
N2O (11). The results are shown in Fig. 3, which includes a
difference spectrum obtained by subtracting the spectrum
under N2O (where only triplet states are observed) from that
recorded under N2 (where both triplet and hydrated electrons
are monitored). The difference spectrum is in full agreement
with that expected for the solvated electron (12).
of FA reported in methanol 5
hydrogen abstraction from the solvent occurs readily (13).
The ketyl radicals have a lifetime of ca. 140 s under our
␮s after the laser pulse, when
␮
conditions and decay by mixed first–second order kinetics.
The trace in the inset of Fig. 1 is analyzed with the sum
of two exponentials. The fitting gives rate constants of 2.05
Ϫ
1
Ϫ1
ϫ
106 s (triplet decay) and 8.9
ϫ
105 s (ketyl growth).
From the latter number and the concentration of FA (3.5
ϫ
Ϫ5
The triplet state assignment is not as straightforward as
that for the hydrated electron; i.e. 360 and 460 nm are not
the regions for the typical absorption of substituted benzo-
10 M), we estimate that the hydrated electron reacts with
Ϫ
1
Ϫ1
FA with a rate constant of 2.6
ϫ
1010
M
s , a value in
line with others for aromatic ketones (14–18). Interestingly,
the presence of a negative charge (i.e. the carboxylate) does
not cause any significant slowdown in the electron capture
reaction. We note that the actual value may be slightly lower
than our estimate if the electron decays by other pathways
in addition to trapping by FA. We also observe that in ad-
dition to the transients discussed previously, there is a con-
siderable bleaching at 300 nm (see inset of Fig. 3). This
indicates that the processes taking place (including triplet
decay) lead to irreversible chemical changes.
In order to determine the origin of the various transients
discussed, we also carried out power dependence studies.
These measurements are normally used to differentiate mon-
ophotonic and multiphotonic processes. In a qualitative ex-
periment, we used 355 nm excitation; the laser available for
these experiments has considerably lower peak power than
the excimer laser used for 308 nm excitation. Under these
conditions, the experiment under nitrogen gave a spectrum
virtually identical to that obtained with 308 nm under N2O,
clearly showing that hydrated electrons were not produced
under these conditions (data not shown).
phenones. In fact, for FA we have reported
␭
max
values of
340 and 520 nm for its triplet state in methanol (13). To test
the hypothesis that the 360 and 460 nm signals are caused
by the triplet state, we carried out quenching experiments
with two selective triplet quenchers, sorbic acid and 1-NM,
and obtained quenching rate constants of 1.9
ϫ
ϫ
109 and 2.9
109 M s , respectively. Further, in the case of 1-NM,
Ϫ
1
Ϫ1
the decay of the FA triplet (monitored at 480 nm) occurs
concurrently with the growth of the 420 nm signal charac-
teristic of the triplet state of 1-NM (see Fig. 4).
Consistent with our triplet state and hydrated electron as-
signments, both signals are quenched by oxygen. The triplet
signal at 340 nm now decays with a rate constant of 5.9
ϫ
Ϫ
1
Ϫ1
106 s , compared with ca. 2
ϫ
106 s under nitrogen or
N2O. From the difference, we estimate the quenching rate
Ϫ
1
Ϫ1
constant for oxygen at 4
expected for these reactions.
ϫ
109 M s , a value in the range
We now turn our attention to the growth observed at 340
nm. As already indicated, the presence of a valley in the
time profile requires that either the growth has a different
precursor (i.e. other than the triplet state) or that one or more
intermediates are involved in the reaction path. Further,
In a separate experiment, we determined the power de-
pendence at 308 nm for three separate transients: the electron

196 Gonzalo Cosa et al.
5
Figure 5. Power dependence of 3.5
ϫ
10Ϫ M FA in 0.01 M phos-
phate buffer solution of pH 7.4; laser at 308 nm; N2 bubbled. The
full dose corresponds to ca. 25 mJ per pulse. Taken at ( ) 340 nm,
3.5 s after the laser pulse; ( ) 460 nm, immediately after the laser
pulse; and ( ) 720 nm, immediately after the laser pulse and cor-
ⅷ
Figure 6. Normalized transient absorption spectrum of 0.035 mM
FB under N2 atmosphere in different acetonitrile:water mixtures with
10 mM NaOH; only half of the experimental points are shown for
␮
Ⅺ
⌬
rected for triplet contribution.
clarity. Laser wavelength 308 nm. (
after the laser pulse; ( ) 4:6 acetonitrile:water, 640 ns after the laser
pulse; ( ) 6.5:3.5 acetonitrile:water, 640 ns after the laser pulse; (⌬)
Ⅵ) 2:8 acetonitrile:water, 320 ns
⅜
ⅷ
8:2 acetonitrile:water, 320 ns after the laser pulse; and (
acetonitrile, 640 ns after the laser pulse (no NaOH).
ࡗ) pure
(720 nm) and the triplet state (460 nm) immediately after
the laser pulse and the ketyl radical (340 nm) after its growth
was complete (see inset of Fig. 1). The plot shown in Fig.
5 clearly shows a quadratic dependence for the electron and
the ketyl radical (fitted with a parabola) and a linear depen-
dence, as expected, for the triplet state. This shows that elec-
tron ejection (and as a consequence ketyl radical formation)
is dominantly (and probably exclusively) the result of two-
photon ionization.
We have attributed the triplet signals to triplet FA. How-
ever, two questions remain. Conceivably, the triplet state ob-
served could be that of the carbanion produced after FA
photodecarboxylation; further, the lifetime of the triplet (0.5
tonitrile:water mixture, and the longer-lived FB triplet was
used as a model.
Figure 7 shows quenching plots in both solvents. The sec-
ond order rate constant (anticipated to be owing to hydrogen
abstraction) corresponds to the slope of these plots. The rate
Ϫ
Ϫ
constant is 1.3
ϫ
108 M 1 s 1 in the acetonitrile-rich solvent,
which compares well with the value of 2.9
ϫ
108 for ben-
zophenone itself in acetonitrile (21). In contrast, in the wa-
ter-rich solvent we see no quenching, yielding a conservative
Ϫ
1
Ϫ1
upper limit of ca. 1
ϫ
107 M
s
for the quenching rate
␮s) is rather short for a substituted benzophenone in water
constant. The observation is consistent with a change in the
(19), suggesting that reactive pathways play a key role in
determining the triplet lifetime.
nature of the excited triplet from n,
(water).
␲* (acetonitrile) to ␲,␲*
The ester, FB, is a good control compound because it does
not undergo the decarboxylation–rearrangement that char-
acterizes the photolysis of the FA anion (6,20). In order to
ensure solvent compatibility, it was necessary to carry out
the experiments in acetonitrile:water mixtures. Figure 6
shows the spectra for FB in various solvent mixtures; base
has been added to match the conditions used with FA in
organic solvents (vide infra). Clearly, FB undergoes the
same shifts as FA in water, thus supporting the assignment
of the early transient of Fig. 1 to triplet FA. However, the
triplet lifetime of FB is much longer than that of FA. For
example, in 8:2 acetonitrile:water, the FB triplet lives 3.80
The temperature dependence of the decay of triplet FA
was also examined in 93:7 acetonitrile:water in the presence
of TMAOH and in water at pH 7.4. These plots are shown
in Fig. 8 and yield log(A/s ) values of 12.7 and 11.9 for
the aqueous and acetonitrile rich solutions; the correspond-
ing activation energies are 36.9 and 23.4 kJ/mol, respective-
ly.
Ϫ
1
The triplet energy for FA was determined in a glass at 77
K from the maximum of the lowest energy band of the phos-
␮s, thus supporting a reactive decay pathway for FA. The
FB triplet lifetime does not change significantly in different
acetonitrile:water mixtures. In the case of FA in the presence
of base (TMAOH), the lifetime decreases as the fraction of
organic solvent in the mixture increases and is 0.025 ␮s in
93:7 acetonitrile:water, about 20 times shorter than in water
at pH 7.4. Studies on the quantum yield of photodegradation
of FA (vide infra) clearly show that the excited state decar-
boxylation is responsible for the fast triplet decay in the case
of the FA anion.
Given the large difference between the FA triplet lifetimes
in aqueous systems at pH 7.4 and in basic 93:7 acetonitrile:
water, we decided to explore the reactivity of the triplet with
1,4-cyclohexadiene, a good hydrogen donor. In order to en-
sure solvent compatibility, water was replaced by a 2:8 ace-
Figure 7. Rate of decay of the triplet state of FB measured at 700
nm in the presence of different amounts of 1,4-cyclohexadiene. (
ⅷ)
0.06 mM FB, 93:7 acetonitrile:water, N2 bubbled and ( ) saturated
⌬
solution of FB in 2:8 acetonitrile:water, N2 bubbled. Laser wave-
length 308 nm.

Photochemistry and Photobiology, 2002, 75(3) 197
Figure 8. Arrhenius plot for FA. (
trile:water, under N2 atmosphere, 0.08 mM FA, 3.4 mM TMAOH.
) Determined in 0.1 M buffer of pH 7.4, under N2O atmosphere,
ⅷ) Determined in 93:7 acetoni-
Figure 9. Normalized phosphorescence spectra of FA in (
ter, saturated solution, exc 310 nm; ( —) 2:1 ethylene glycol–water,
0.2 mM, 314 nm; and ( ---) 4:1 ethanol–methanol, 0.08 mM,
305 nm. Also shown is ( ---) the spectrum of FB in 0.1 mM
288 nm.
Ⅺ---) wa-
(⅜
␭
⅜
0.07 mM FA. Laser wavelength 308 nm.
␭
ࡗ
⌬
exc
␭
exc
methylcyclohexane,
␭
exc
phorescence spectra (i.e. the 0-0 band) shown in Fig. 9. The
data are summarized in Table 1 and clearly show that a
decrease in triplet energy accompanies the increase in water
content, for a total triplet energy change of 13 kJ/mol. These
changes in triplet energy were also accompanied by changes
in the phosphorescence lifetime (longer in higher polarity
amounts of FA dimers originating from the FA ketyl radical
were also detected after irradiation with the 308 nm laser.
The quantum yield (⌽) for FA photodegradation was de-
termined after steady state irradiation of FA in 93:7 aceto-
nitrile:water and water under basic conditions and using ke-
toprofen as a standard; the results are presented in Fig. 10.
In both cases, photoproducts 1 and 2 were the only species
formed. The ratio of compound 1 to compound 2 in aqueous
solutions is 69:31 (slightly different from that previously re-
ported, i.e. 78:22 [23]) and that in 93:7 acetonitrile:water is
1:1. The product 2 is rationalized by a Wittig rearrangement
of the carbanion, which is disfavored in more polar solvents;
this results in the observed change in product ratio with sol-
vent composition, in agreement with previous results in
phosphate buffer solution, acetone and methanol (23).
The preceding results lead us to propose a reaction scheme
for FA similar to that encountered in methanol solutions.
The general mechanism shown in Scheme 1 seems to apply
to the FA anion photochemistry in all the conditions em-
ployed, although with different efficiencies for each solvent
system.
glasses), also consistent with a switch toward a ␲,␲* char-
acter (22). We note this evolution may be more complete in
solution at room temperature, where solvation may be more
extensive and where the species is the carboxylate anion, as
opposed to the acid at 77 K in the experiments of Table 1.
Product studies were performed using as the excitation
source a 355 and a 308 nm laser. A total of 100 shots was
delivered on each 2 mL sample, and 10 samples were col-
lected. The samples exposed to a given wavelength were
then combined and extracted with dichloromethane. After
solvent evaporation, the samples were injected on a GC/MS
instrument. The same procedure was followed with a non-
irradiated sample for comparison. From the analysis of the
mass spectra corresponding to each of the peaks in the chro-
matogram, we were able to identify 4-chloro-4
benzophenone (1) and 4-chloro-4 -(1-hydroxy-1-methyle-
Ј-isopropoxy
Ј
thyl) benzophenone (2) (see Scheme 1), in agreement with
the results previously obtained in methanol solutions (13).
No difference in product distribution was found in using
different lasers as the excitation source.
DISCUSSION
The analysis of photoproducts was also done in an HPLC
provided with a reverse phase column. The same product
ratio was obtained for compounds 1 and 2 when FA was
irradiated either at 355 or 308 nm in buffer solution. Minor
There are several differences between the photobehavior of
FA in aqueous solutions and in organic polar solvents such
as methanol, where its photochemistry is already well un-
derstood (13). One of these differences relates to the occur-
Table 1. Solvent effects on the triplet energy of FA at 77 K. Also shown are the triplet decay rate constants and their respective weights
Triplet
energy
(kJ/mol)
1
1
Solvent
0,0 Band (nm)
k1 (sϪ
)
A1
k2 (sϪ
)
A2
Methylcyclohexane*
4:1 Methanol:ethanol
2:1 Ethylene glycol:water
Water
418
417
423
437
287
287
283
274
144
103
29
1
1
0.47
0.54
11
10
0.53
0.46
51
*Solution of fenofibrate was employed. Excitation wavelengths employed were 288 nm for FB in methylcylcohexane, 305 nm for FA in
methanol:ethanol, 310 nm for FA in water and 314 nm for FA in ethylene glycol:water.

198 Gonzalo Cosa et al.
Small transient absorption extending from 330 to 420 nm
is observed at very long time scales. This may be caused by
either a cyclohexadienyl radical proposed as a product from
the benzophenone cation after photoionization (27) or by the
light absorbing transients that participate in the rearrange-
ment of FA to product 2 in Scheme 1 (13). A detailed in-
terpretation of these minor signals is not essential for un-
derstanding the details of the mechanism.
The other key transient observed in this work is the triplet
state of FA, which, not surprisingly, results from a mono-
photonic process. This transient will be important under con-
ditions of laser or ambient illumination. Product studies
show that the fast triplet decay reflects decarboxylation, ul-
timately leading to products 1 and 2.
Several aspects of triplet state spectroscopy and lifetimes
(see Tables 1 and 2) need to be considered together: (1) the
triplet lifetime of the FA anion is much shorter than for FB;
(2) the triplet lifetime of the anion form of FA is 10 and 20
times shorter in methanol and acetonitrile-rich solvents, re-
spectively, than in water; (3) the quantum yield of FA pho-
todegradation is ca. 12 times larger in acetonitrile-rich so-
lutions than in water; (4) the triplet–triplet absorption of FA
in water no longer resembles the typical benzophenone trip-
let structure observed in methanol (13), i.e. the visible max-
imum is at 460 nm rather than at 525 nm; (5) the triplet state
of FB (a model for the FA triplet state) reacts readily with
hydrogen donors in organic solvents but is at least 20 times
less reactive in water; and (6) the phosphorescence spectra
and lifetimes at 77 K suggest a change in the lower triplet
Scheme 1. Photodegration mechanism for FA.
rence of photoionization processes; it is not unusual for these
processes to be enhanced in aqueous media. Further, in the
case of FA at pH 7.4, where it is present as the carboxylate,
photoionization does not need to overcome electrostatic at-
tractions in order to eject the electron (as is the case for
neutral species). Similar processes have been reported for
benzophenone and ketoprofen (15,24–26).
Hydrated electrons decay predominantly by addition to the
ground state of FA with a rate constant value of around 2.6
excited state from n,
The six issues raised in the previous paragraph can be
easily rationalized by invoking a * triplet in water for
␲* in organic solvents to ␲,␲* in water.
Ϫ
1
Ϫ1
ϫ
1010
M
s . Interestingly, the rate is just what it would
be if no negative charge were present in the substrate. Sim-
ilar observations were made in the case of the 3,3
benzophenonetetracarboxylate ion (16).
␲,␲
Ј,4,4Ј-
FA. Such state inversion is common for acetophenone and
xanthone derivatives but less so for benzophenones, which
are usually the archetypes for n,␲* behavior, although sin-
glet photochemistry has been reported recently for ketopro-
fen, another substituted benzophenone (24,25).
The spectral shift observed for triplet FA is in line with
those observed for xanthone and azaxanthone (9,28). The
band in the T-T absorption spectrum of FB or FA is 34 kJ/
mol higher in energy in water (460 nm) than in acetonitrile
(525 nm).
We note that the spectrum observed after electron trapping
by FA is clearly that of the ketyl radical, rather than that of
the benzophenone radical anion. This is not unexpected be-
cause the pKa of the ketyl radical from benzophenone is 9.25
(19), and thus, rapid protonation should occur at pH 7.4.
Power dependence studies show that electron photoejec-
tion and the resulting ketyl radical formation are predomi-
nantly (and presumably exclusively) two-photon processes.
The implication of this is that from a biological point of view
these processes are not important because they will not occur
under conditions of ambient (solar or artificial) illumination
(4).
An interesting observation from this work is that decar-
boxylation, which in the case of FA involves the triplet state,
is much faster for the n,␲* than for the
␲,␲* triplet, and
Table 2. Photophysical and photochemical parameters obtained for FA and FB in different acetonitrile:water mixtures
Ratio acetonitrile:water
Compound
93:7
0.70
8:2
4:6
2:8
0:1
⌽
Photodegradation*
FA
FA
FB
FB
FB
0.06
kdec (sϪ1)†
3.9
2.71
ϫ
ϫ
107
2.4
2.64
ϫ
ϫ
107
105
5.3
3.10
ϫ
ϫ
106
105
2.05 ϫ
106
kdec (sϪ1)‡
105
2.54
ϫ
460
ϫ
105
T-T absorption (nm)
530
ϫ
1
kq 1,4-CHD (MϪ sϪ1)§
3.0
108
Յ1
107
*Values obtained with 80 mM TMAOH.
†The solution was prepared with 8 mM TMAOH; the rate constants were measured at 650 nm.
‡The rate constants were measured at 450 nm.
§The rate constants were measured at 700 nm.

Photochemistry and Photobiology, 2002, 75(3) 199
Figure 11. Energy level diagram for the lowest triplet state (T1) of
FA in water and acetonitrile, the second lowest triplet state (T2) of
FA in water and the higher triplet state level giving rise to the T1-
Tn absorption band, where A is probably in the 5 to 15 kJ/mol range
and B (based on the spectral shift for T-T absorption) is likely to
be somewhat higher.
Figure 10. A: Concentration of FA after different irradiation dura-
tions with a 254 nm Hg lamp under N2 atmosphere, 0.08 M TMAOH
in (
evolution of ketoprofen concentration in water. B: Product formation
over time, following FA irradiation. ( ) photoproduct 1, water; (
photoproduct 2, water; ( ) photoproduct 1, 93:7 acetonitrile:water;
) photoproduct 2, 93:7 acetonitrile:water. The concentrations were
measured by HPLC.
Ⅵ) water and (Ⅺ) 93:7 acetonitrile:water; also shown is (ࡗ) the
(i.e. 308 and 355 nm excitation wavelength) in aqueous buff-
er solutions. In its dissociated form, FA decarboxylates from
the triplet manifold after excitation, in a way similar to that
previously reported in alcohol mixtures in the presence of
base (13). This deactivation mode is inactive when FA is in
its ester form (FB). In the latter case, the triplet state decays
with a similar rate, independent of the solvent media; how-
ever, in the presence of good hydrogen donors hydrogen
abstraction is faster in lower polarity acetonitrile:water mix-
tures, compared with pure water. This is in parallel with a
higher reactivity toward decarboxylation for the FA anion in
acetonitrile-rich mixtures, compared with water. This in turn
correlates with a higher triplet energy in ethanol vs water
and is also accompanied with a hypsochromic shift in the
visible T-T absorption band, compared with that in aceto-
nitrile, similar to the behavior of xanthone (9) or azaxantho-
ne (28). These results are explained in terms of FA present-
᭡
ⅷ)
᭝
(⅜
thus, triplet lifetimes are longer in water than in organic
solvents (similar results have been obtained for tiaprofenic
acid) (29). At least part of this slowdown is reflected in the
activation energy. Note for example in Fig. 8 that the lower
line clearly has the greater slope, even if differences in
preexponential factors are probably within experimental er-
ror, given the limited temperature range available.
If decarboxylation occurs from the triplet state of FA, then
it is reasonable to assume that at least the nascent carbanion
must be in the triplet state. In Scheme 1 we have written the
carbanion and the biradical as resonant structures. Whereas
singlet and triplet states should be essentially isoenergetic in
the biradical case, large differences in energy are anticipated
for the carbanion. Thus, the carbanion should be the pre-
ferred resonance structure for the singlet, whereas in the trip-
let manifold the biradical will be dominant. For the biradical
to be the nascent species, an electron transfer (carboxylate
to carbonyl) may precede decarboxylation.
ing a
low polarity acetonitrile:water mixtures the lowest triplet is
higher in energy and has an n, * character (see Fig. 11).
Both decarboxylation and hydrogen abstraction are process-
es arising from the n, * state.
␲,␲* nature lowest triplet state in water, whereas in
␲
␲
From a biological point of view, these results are of sig-
nificance. We can now explain the lower reactivity exhibited
by FA and its photoproducts when compared with ketopro-
fen and its respective photoproducts. In the latter case it has
been established that the deleterious effects to cell mem-
branes arise from lipid peroxidation. This is a radical-me-
Phosphorescence studies support the foregoing analysis,
i.e. increase of aqueous content leads to a decrease in triplet
energy and an increase in triplet lifetime. However, the
switch to a ␲,␲* state with aqueous content is probably more
complete at room temperature than in a glass at 77 K be-
cause in the latter the phosphorescence spectrum shows
some vibrational resolution in water, whereas at room tem-
perature the unreactivity of the triplet toward hydrogen
transfer (see Fig. 7) suggests a complete inversion of states.
In conclusion, in this work we have presented results on
the photochemistry of FA both at high and low laser power
diated mechanism involving the action of an n,␲* triplet ke-
tone acting as an initiator (30–32). Photoproduct 1, whose
structure and excited state spectroscopic behavior after LFP
is similar to that of FB (data not shown), should also present
a
␲,␲* lower triplet state in water, therefore being less re-
active toward lipid peroxidation at physiological pH values.

200 Gonzalo Cosa et al.
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cleavage. As has been shown, this is a process inhibited by
radical quenchers and is therefore most probably initiated
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␲
* lowest triplet states being more reactive than
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Acknowledgements J.C.S. thanks the Natural Sciences and Engi-
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research grant. G.C. thanks the Ontario Graduate Scholarship Pro-
gram for a Post-Graduate scholarship. M.A.M and F.B. wish to ac-
knowledge the financial support from the Generalitat Valenciana
(grant GV.DOC98-VS-24-2) and the European Union (grant BMH-
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