Photobehavior of mixed π*/ππ* triplets: Simultaneous detection of the two transients, solvent-dependent hydrogen abstraction, and reequilibration upon protein binding

Article abstract of DOI:10.1021/jp2051432

In the present work, a systematic study on hydrogen abstraction by the excited triplet states of 4-methoxybenzophenone (1) and 4,4′- dimethoxybenzophenone (2) from 1,4-cyclohexadiene (3), 4-methylphenol (4), 1,2,3,4-tetrahydroquinoline (5), and 1-methyl-1,2,3,4-tetrahydroquinoline (6) in different media has been undertaken. Laser flash photolysis (LFP) revealed that in nonpolar solvents, 1 and 2 triplets have a π* configuration with the typical benzophenone-like T-T absorption spectrum (?_max ca. 525 nm). Conversely, in aqueous solution, transient absorption spectra with maxima at 450 and 680 nm, attributed to the ππ* triplet, were obtained. Quenching of 1 or 2 triplet by 3 led to ketyl radical formation with rate constants in the range of 10⁶-10⁸ M^-1 s ^-1, being one order of magnitude higher in acetonitrile than in aqueous media. The rate constants of quenching by 4 and 5 were similar in both polar and nonpolar solvents; the highest value was found for 6 in acetonitrile ((6.3 to 6.9) - 10⁹ M^-1 s^-1). For mechanistic insight, LFP of 1 or 2 in the presence of dimethoxybenzene as electron donor was performed. The results showed that in this case, triplet quenching is favored in aqueous solution. In addition, 2 included in human serum albumin (HSA) was submitted to LFP. The decay kinetics, monitored at 430 nm, fitted well with three lifetimes of 0.45, 1.4, and 14.4 ?s assignable to 2 in bulk solution and in site II or in site I of HSA, respectively. This assignment was confirmed by using oleic acid and ibuprofen as selective displacement probes.

Full text of DOI:10.1021/jp2051432

ARTICLE
pubs.acs.org/JPCB
Photobehavior of Mixed nπ*/ππ* Triplets: Simultaneous Detection of
the Two Transients, Solvent-Dependent Hydrogen Abstraction, and
Reequilibration upon Protein Binding
Dolors Jornet, Rosa Tormos,* and Miguel A. Miranda*
Departamento de Química/Instituto de Tecnología Química UPV-CSIC, Universidad Politꢀecnica de Valencia, Avenida de los Naranjos
s/n, E-46022 Valencia, Spain
ABSTRACT:
In the present work, a systematic study on hydrogen abstraction by the excited triplet states of 4-methoxybenzophenone (1) and
4,40-dimethoxybenzophenone (2) from 1,4-cyclohexadiene (3), 4-methylphenol (4), 1,2,3,4-tetrahydroquinoline (5), and
1-methyl-1,2,3,4-tetrahydroquinoline (6) in different media has been undertaken. Laser flash photolysis (LFP) revealed that in
nonpolar solvents, 1 and 2 triplets have a nπ* configuration with the typical benzophenone-like T-T absorption spectrum (λmax ca.
525 nm). Conversely, in aqueous solution, transient absorption spectra with maxima at 450 and 680 nm, attributed to the ππ* triplet,
were obtained. Quenching of 1 or 2 triplet by 3 led to ketyl radical formation with rate constants in the range of 106ꢀ108 Mꢀ1 sꢀ1
,
being one order of magnitude higher in acetonitrile than in aqueous media. The rate constants of quenching by 4 and 5 were similar
in both polar and nonpolar solvents; the highest value was found for 6 in acetonitrile ((6.3 to 6.9) ꢁ 109 Mꢀ1 sꢀ1). For mechanistic
insight, LFP of 1 or 2 in the presence of dimethoxybenzene as electron donor was performed. The results showed that in this case,
triplet quenching is favored in aqueous solution. In addition, 2 included in human serum albumin (HSA) was submitted to LFP. The
decay kinetics, monitored at 430 nm, fitted well with three lifetimes of 0.45, 1.4, and 14.4 μs assignable to 2 in bulk solution and in
site II or in site I of HSA, respectively. This assignment was confirmed by using oleic acid and ibuprofen as selective displacement
probes.
’ INTRODUCTION
the formation of the nπ* triplet alone, whereas a nπ*/ππ*
mixture is observed in acetonitrile. Similar studies on 2 in
acetonitrile solution also reveal the presence of an equilibrated
nπ*/ππ* mixture. In aqueous solution, both aromatic ketones
show only their ππ* triplets.5,6
The reactivity of nπ* ketone triplets toward aliphatic or
benzylic hydrogen donors is higher than that presented by ππ*
triplets. Moreover, it is generally accepted that the reactivity is
basically due to nπ* triplet excited states and that ππ* triplets are
essentially unreactive. Furthermore, ππ* ketones are thought to
react through the thermally populated nπ* triplets.4,7,8 There-
fore, in the case of a high energy gap between the two states, the
One of the most deeply studied photoreactions of aromatic
ketones is hydrogen atom abstraction.1 The reactivity of a
carbonyl compound toward a hydrogen donor depends on the
electronic configuration of the lowest triplet excited state (nπ* or
ππ*),2 which may change with solvent polarity and aromatic ring
substitution. Therefore, acetophenone presents a nπ* triplet in
nonpolar solvents and a ππ* triplet in polar solvents; electron-
donating substituents may produce an inversion of the electronic
configuration of the acetophenone triplet.3 By contrast, the
benzophenone triplet is nπ* in both polar and nonpolar
solvents;4 inversion of the configuration from nπ* to ππ* is
favored by increasing solvent polarity, as in 4-methoxybenzo-
phenone (1) and 4,40-dimethoxybenzophenone (2), where the
aromatic ring bears electron-donating substituents.5 Therefore,
laser flash photolysis (LFP) of cyclohexane solutions of 1 shows
Received: June 1, 2011
Revised:
July 22, 2011
Published: July 26, 2011
r
2011 American Chemical Society
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ARTICLE
Chart 1. Chemical Structures of Ketones and Donors
presence of potassium carbonate.23 The purity of all products was
checked by HPLC and H NMR. Reagent-grade solvent acet-
1
onitrile was purchased from Scharlau and used without further
purification. The neutral buffer PBS (0.01 M phosphate buffer,
0.0027 M potassium chloride, and 0.137 M sodium chloride) was
purchased from Sigma.
Absorption Spectra. Optical spectra in different solvents
were measured on a Perkin-Elmer Lambda 35 UV/vis spectro-
photometer.
Laser Flash Photolysis Experiments. The LFP experiments
were carried out by using a Q-switched Nd/YAG laser (Quantel
Brilliant, 355 nm, 14 mJ per pulse, 5 ns fwhm) coupled to
an mLFP-111 Luzchem miniaturized equipment. This transient
absorption spectrometer includes a ceramic xenon light source,
125 mm monochromator, Tektronix 9-bit digitizer TDS-3000
series with 300 MHz bandwidth, compact photomultiplier and
power supply, cell holder and fiber optic connectors, fiber optic
sensor for laser-sensing pretrigger signal, computer interfaces,
and a software package developed in the LabVIEW environment
from National Instruments. The LFP equipment supplies 5 V
trigger pulses with programmable frequency and delay. The rise
time of the detector/digitizer is ∼3 ns (2.5 GHz sampling). The
laser pulse is probed by a fiber that synchronizes the LFP system
with the digitizer operating in the pretrigger mode. All transient
spectra were recorded using 10 ꢁ 10 mm2 quartz cells with 4 mL
of capacity, and all were bubbled during 20 min with N2.
Absorbance of the samples was kept between 0.2 and 0.3 at the
laser wavelength. All experiments were carried out at room
temperature.
ketone is typically unreactive. However, if an electron transfer
step precedes (or is coupled to) hydrogen transfer, then the
electronic configuration of the lowest triplet state becomes
unimportant.9
In addition, the XꢀH bond energy and the oxidation potential
of the donor have a strong influence on formal hydrogen abstrac-
tion. Therefore, as a function of the above parameters, the process
can occur by a variety of mechanisms, ranging from pure hydrogen
atom abstraction (hydrocarbons or alcohols)3,10 to electron
transfer, followed by proton transfer from the initially formed
radical cation to the carbonyl radical anion (amines).11ꢀ14 Be-
tween these two extreme situations, an intermediate behavior can
be found in benzylic or phenolic derivatives. In the former, the
involvement of a charge-transfer complex has been reported,9,15
whereas for the latter, coupled electronꢀproton transfer within
hydrogen bonded exciplexes has been suggested.16ꢀ20
In this context, the aim of the present work is to perform a
systematic study on the triplet reactivity of 4-methoxybenzophe-
none (1) and 4,40-dimethoxybenzophenone (2) in the presence
of different types of hydrogen donors (Chart 1) using the LFP
technique. Our primary interest is (i) to establish the conditions
for simultaneous detection of the two types of triplets, (ii) to
determine whether for the same donor the change in the medium
polarity may have an influence on the reactivity through triplet
inversion, and (iii) to compare for each type of ketone triplet the
reactivity changes as a function of the hydrogen donor nature.
In addition, as a paradigm to study the influence of constrained
microenvironment on the nature and behavior of the triplet
excited state, aromatic ketone 2 has been included in human
serum albumin (HSA). This protein contains different binding
sites with distinct properties; its two high affinity sites for small
heterocyclic or aromatic compounds are known as site I and site
II. In the former, hydrophobic interactions predominate, whereas
in the latter, binding is governed by hydrogen bonding and
electrostatic interactions.21 The properties of triplet excited
states are very sensitive to the intraprotein microenvironment.22
Therefore, analysis of the triplet decay can provide information
about substrate distribution among the bulk solution and the
protein binding sites, whereas changes in the spectral shape are
expected to report on the possible reequilibration between nπ*
and ππ* triplets.
’ RESULTS AND DISCUSSION
Dynamic studies on the photoreaction of 1 and 2 with 3ꢀ7
were performed in cyclohexane, acetonitrile, and mixtures of
acetonitrile and buffered aqueous solution using 355 nm laser
excitation (Nd/YAG).
In cyclohexane solution, LFP of 1 afforded a transient with
a spectrum similar to that displayed by benzophenone
(Figure 1A). However, in deareated acetonitrile solution, LFP
of 1 led to transients absorbing in the 300ꢀ700 nm range. The
spectrum showed a broad band centered at 520 nm and a
shoulder at 450 nm, assigned to the nπ* and ππ* triplet,
respectively (Figure 1B).5 Under the same conditions, 2 pre-
sented two bands centered at 540 and 425 nm. The former
matched with the previously reported nπ* triplet,19 whereas the
latter (more important in acetonitrile) can be assigned to the ππ*
triplet (Figure 1A,B).
When LFP of 1 and 2 was performed in a mixture of PBS and
acetonitrile (3:1 v/v), the obtained spectra, with maximum at
450 nm and significant absorption in the 600ꢀ700 nm region,
corresponded to the ππ* triplet of both aromatic ketones
(Figure 1C). In the case of 1, the triplet energy in water was
previously estimated to be ca. 13 kJ molꢀ1 lower than that in
cyclohexane (288 kJ molꢀ1).5 However, for 2, it was assumed in
previous studies that the lowest triplet is basically nπ*,3,9 and its
energy was estimated from the phosphorescence 0,0 band at 77 K
in methyltetrahydrofuran as 290 kJ molꢀ1.9 This triplet energy
value has also been assumed in acetonitrile solution.19b To
determine in a systematic way the energy of the two closely
lying triplet states of 2 in solution at room temperature,
quenching experiments with several potential energy acceptors
were undertaken. Therefore, it was expected that the most
’ EXPERIMENTAL SECTION
Materials and Solvents. 4-Methoxybenzophenone (1), 4,40-
dimethoxybenzophenone (2), 1,4-cyclohexadiene (3), 4-methyl-
phenol (4), 1,2,3,4-tetrahydroquinoline (5), 1,4-dimethoxyben-
zene (7), 1,3-cyclohexadiene, biphenyl, fluorene, dibenzofuran,
toluene, 2-propanol, and HSA were purchased from Aldrich.
1-Methyl-1,2,3,4-tetrahydroquinoline (6) was synthesized by
treatment of an acetone solution of 5 with methyl iodide in the
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ARTICLE
Table 1. Rate Constants for Energy Transfer from 4,40-
Dimethoxybenzophenone to 1,3-Cyclohexadiene, Biphenyl,
Fluorene, and Dibenzofuran, Together with the Estimated
Values of the Triplet Excited State Energies
a
kET (Mꢀ1 sꢀ1
)
ET (kJ molꢀ1
)
PBS/acetonitrile
toluene
1,3-cyclohexadiene
biphenyl
219b
271b
282b
289e
4.0 ꢁ 109
4.9 ꢁ 109
4.6 ꢁ 109
2.8 ꢁ 108
4.3 ꢁ 109
4.6 ꢁ 109
3.1 ꢁ 109
8.9 ꢁ 108
fluorene
dibenzofuranc,d
a Obtained using 2 ꢁ 10ꢀ5 to 1.5 ꢁ 10ꢀ3 M concentrations of the
quenchers under N2. b Value in nonpolar solvent.24 c ΔET = 7 kJ molꢀ1
in PBS/acetonitrile. d ΔET = 3 kJ molꢀ1 in toluene. e Value in ethyl
acetate.25
Therefore, LFP of 2 in PBS/MeCN was performed at 355 nm
in the presence of increasing amounts of quenchers, and the
decay of the T-T band was analyzed at 430 nm to determine
the quenching rate constants. When the reciprocal triplet life-
times of 2 were plotted against the quencher concentrations,
different straight lines were obtained (not shown). The slopes
(intermolecular kET values) are given in Table 1, together with
the triplet energies of the acceptors, required for application of
the Sandros’ equation and assuming that the optimum rate
constant for the system (kmax) is 4.5 ꢁ 109 Mꢀ1 sꢀ1, the average
of the first two entries. Therefore, a reasonable estimation for the
ππ* triplet energy of 2 is 282 kJ molꢀ1. In a similar way,
quenching experiments in toluene solution (λmax = 355 nm),
where kmax was taken as 4.4 ꢁ 109 Mꢀ1 sꢀ1, led to a nπ* triplet
energy of 286 kJ molꢀ1 (Scheme 1). A simplified energy diagram
for the triplet excited states of 1 and 2 in different solvents is
shown in Scheme 1.
After characterization of the two triplets of 2, experiments on
hydrogen abstraction were undertaken. The results are summar-
ized in Table 2. When 1,4-cyclohexadiene (3) was used as donor,
the ratio between the quenching rate constants measured in
acetonitrile and PBS/MeCN (3:1 v/v) resulted to be ca. 30
and 13 for 1 and 2, respectively. In a previous study on the
photophysical properties of 1, it was shown that an increase in the
polarity of the medium produces a decrease in ππ* triplet energy,
without affecting the nπ* triplet.5 LFP of 5.0 ꢁ 10ꢀ4 M solutions
of 1 or 2 in different mixtures of PBS and acetonitrile in the
presence of 3 (2.5 ꢁ 10ꢀ3 to 5 ꢁ 10ꢀ2 M) were carried out to
analyze the influence of solvent polarity on the effectiveness of
hydrogen abstraction by the excited ketones. Therefore, Figure 2
shows the dependence of the rate constant on the molar fraction
of acetonitrile, where it can be clearly seen that the kinetics
become faster in enriched acetonitrile solutions.
Figure 1. Normalized transient absorption spectra obtained by 355 nm
laser photolysis of 4-methoxybenzophenone (b, red) and 4,40-di-
methoxybenzophenone (9) 0.12 μs after the pulse in (A) cyclohexane,
(B) MeCN, and (C) PBS/MeCN (3:1, v/v).
As mentioned above, carbonyl compounds with lowest-lying
ππ* triplets abstract H atoms from hydrocarbons much more
slowly than their analogs having similar excitation energies but
with the lowest nπ* triplets. In addition, it has been postulated
that ππ* triplets abstract hydrogen atoms predominantly via
their thermally populated, higher energy nπ* states. In this
context, temperature changes should modify the reactivity. To
check this point, we performed LFP experiments with 2 in
toluene and PBS/MeCN solutions in the presence of 2-propanol
as hydrogen donor, at temperatures ranging between 5 and 65 C°
and using 550 or 430 nm, respectively, as the monitoring
stable triplet in toluene is nπ*, whereas in polar medium
(waterꢀMeCN mixtures), the ππ* triplet should be lower in
energy. Therefore, 1,3-cyclohexadiene,24 biphenyl,24 fluorene,24
and dibenzofuran25 were selected as triplet quenchers.
The rate constant of tripletꢀtriplet energy transfer (kET
and the acceptor, as given by Sandros’ eq 126
)
depends on the triplet energy gap (ΔET) between the donor
kET ¼ ðkmax expð ꢀ ΔET=RTÞÞ=ðexpð ꢀ ΔET=RTÞ þ 1Þ
where kmax is the optimum rate constant for the system.
ð1Þ
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ARTICLE
Scheme 1. Energetic Diagram for the Lowest Triplet States
of 1 (ꢀ, blue) and 2 (ꢀ, red) in Solvents of Different Polarity
Table 2. Rate Constants for Quenching of 1 and 2 Excited
Triplet States by Donors
Figure 2. Plot of the reaction rate constants of 1,4-cyclohexadiene with
1 (b, red) and 2 (9) versus molar fraction of MeCN in MeCN/PBS
mixtures.
kq (Mꢀ1 sꢀ1) for (1)
kq (Mꢀ1 sꢀ1) for (2)
PBS/
PBS/
MeCN
MeCN
MeCN
MeCN
550 nm
450 nm
520 nm
440 nm
430 nm
quencher (ππ*)
(nπ*)
(ππ*)
(ππ*)
(nπ*)
3
4
5
6
7
2.8 ꢁ 106 7.5 ꢁ 107 3.8 ꢁ 106 5.2 ꢁ 107
3.1 ꢁ 109 1.2 ꢁ 109 3.9 ꢁ 109 1.3 ꢁ 109
4.8 ꢁ 107
1.0 ꢁ 109
5.5 ꢁ 109 1.5 ꢁ 1010 7.1 ꢁ 109 1.4 ꢁ 1010 1.5 ꢁ 1010
3.9 ꢁ 109 6.3 ꢁ 109 6.3 ꢁ 109 6.4 ꢁ 109
6.9 ꢁ 109
9.5 ꢁ 108 7.2 ꢁ 107 3.7 ꢁ 109 6.9 ꢁ 107 6.9 ꢁ 107
wavelength. The donor selection was based on its low volatility
compared with 1,4-cyclohexadiene, which should minimize con-
centration changes at higher temperatures. The obtained data
actually showed that the quenching constant increases with
temperature, and those changes were more pronounced in polar
solvent. The Arrhenius plot (Figure 3) provided the activation
energy Ea and the pre-exponential factor for 2 in the two solvents.
Therefore, Ea in toluene resulted to be ca. 8 kJ molꢀ1, whereas in
PBS/MeCN, it was estimated at 21 kJ molꢀ1. Hence, it is possible
to correlate the activation energy for hydrogen abstraction with
the energy gap between the two states, provided that the intrinsic
activation energy for the reaction of the nπ* triplets is also taken
into account.
Figure 3. Arrhenius plots for the reaction of triplet excited 2 with
2-propanol in toluene (9) and PBS/MeCN (3:1 v/v) (b, red).
Table 3. Free-Energy Changes for the Intermolecular Elec-
tron Transfer and Exciplex Formation in Acetonitrile Using
the Rehm-Weller Relationshipsa
When 4-methylphenol (4) was used as hydrogen donor, the
quenching rate constants for 1 and 2 in acetonitrile were found to
ketone 1
ketone 2
be (1.2 and 1.0) ꢁ 109 Mꢀ1
s
ꢀ1, respectively, more than one
quencher
Eox (V)
order of magnitude higher that those obtained for 3 ((7.5 and
4.8) ꢁ 107 Mꢀ1sꢀ1) under the same conditions (Table 2). In
aqueous medium, these differences were even more pronounced
(ca. three orders of magnitude). The marked substrate and
solvent dependence of the reaction rate strongly suggest a change
in the reaction mechanism, associated with the involvement of
charge separation.
ΔGet (eV) ΔGex (eV) ΔGet (eV) ΔGex (eV)
4
5
6
1.5419b
0.6031
0.6231
0.44
ꢀ0.49
ꢀ0.48
0.79
ꢀ0.15
ꢀ0.13
0.52
ꢀ0.42
ꢀ0.40
0.87
ꢀ0.07
ꢀ0.05
a Ered(1): ꢀ1.95 V and Ered(2): ꢀ2.016 V versus SCE in MeCN.19b
Similar behavior was observed for 1,2,3,4-tetrahydroquinoline
(5) and 1-methyl-1,2,3,4-tetrahydroquinoline (6). It is well
known that amines are good electron donors and that in their
photoreaction with ketones electron transfer precedes proton
transfer.27
The rate constants for quenching of 1 and 2 by the same donor
in a given solvent were similar. In addition, it is remarkable that
the rate constants for quenching of the nπ* or ππ* triplets of 2 in
MeCN by a selected hydrogen donor were comparable. This is in
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Figure 4. (A) Normalized transient absorption spectrum of 2 in PBS in the absence and in the presence of HSA (1.25 ꢁ 10ꢀ3 M). (B) Laser flash
photolysis (λexc = 355 nm) of 2 (ꢀ) and 2/HSA at different molar ratios 1:0.25 (ꢀ, red) and 1:2.5 (ꢀ, blue). Normalized decays were monitored at
430 nm.
agreement with the fact that the two triplet levels of 2 are strongly
mixed, as expected for a triplet energy gap <8 kJ molꢀ1 4,28
.
By contrast, when the quencher used was 1,4-dimethoxyben-
zene, which can only play the role of an electron donor, the rate
constants for both ketones were more than one order of
magnitude higher in aqueous medium than in organic solvent.
This can be attributed to stabilization of the resulting radical ion
pair by hydration.
The rate constants for reaction of 1 and 2 with 4, 5, and 6 were
high compared with those obtained with 3. The difference can be
explained by facilitation of hydrogen abstraction through exciplex
formation or photoinduced electron transfer. In general, exciplex
formation is favored in nonpolar solvents, whereas photoinduced
electron transfer dominates in polar media.29 The energetics of
radical ion pair formation resulting from electron transfer (et)
can be calculated with eq 230
Figure 5. Contribution (%) of the different lifetime coꢀm4ponents of
the T-T signal of 4,40-dimethoxybenzophenone (5 ꢁ 10 M) in the
different microenvironments: free (gray) and within site I (blue) or site
II (purple) of HSA (1.25 ꢁ 10ꢀ3 M), obtained from the A1, A2, and A3
values of the decay fitting at 430 nm in the presence of different
concentrations of oleic acid.
ꢂ
ΔGet ¼ Eox ꢀ Ered ꢀ E þ ð2:6=ε ꢀ 0:13Þ eV
ð2Þ
where Eox and Ered are the redox potentials (V) of donor and
acceptor, respectively, E* is the excitation energy (eV) of the
involved excited state, and ε is the dielectric constant of the
solvent (37.5 for acetonitrile). Thus, using the values given in
Table 3 for the above-mentioned parameters,19b,31 an exergonic
thermodynamics for electron transfer quenching of the triplet
state of 1 or 2 can be established for 5 and 6, in acetonitrile. By
contrast, the process was found to be endergonic for phenol.
In regards to the thermodynamics of exciplex formation,
calculations according to eq 3,30 where the μ2/F3 value is 0.31
in acetonitrile (where μ the viscosity and F is the polarity solvent
used), indicate that the process is disfavored for all ketone-
quencher pairs. (See Table 3.)
To study the influence of microheterogeneous media on the
nature and behavior of the triplet excited state, we included
ketone 2 in HSA. Aqueous solutions containing 2 and HSA
(molar ratio from 1:0 to 1:5) were prepared in neutral buffer PBS
and submitted to LFP. The transient absorption spectra obtained
after laser excitation (λexc = 355 nm) in the presence of HSA were
similar to that obtained for 2 in acetonitrile. This is shown in
Figure 4A, where the transient spectrum in PBS solution is also
included for comparison. Clearly, a modification of the spectral
shape is observed upon the addition of HSA, pointing to a
marked reequilibration from ππ* to nπ* triplets within the more
lipophilic protein cavities. Moreover, HSA complexation led to
clear changes in the decay traces up to 1:2.5 molar ratios
(Figure 4B).
2
3
ꢂ
ΔGex ¼ Eox ꢀ Ered ꢀ E ꢀ μ =F ½ðε ꢀ 1=2ε þ 1Þ ꢀ 0:19ꢃ þ 0:38 eV
ð3Þ
The decay kinetics, monitored at 430 nm in air-equilibrated
solutions, fitted well with three lifetimes of 0.45, 1.4, and 14.4 μs.
Regression analysis of the decay curves for several 2/HSA ratios
was performed to obtain the preexponential factors A1, A2, and
A3 corresponding to the different lifetimes. The shortest-living
Although all required redox potentials in aqueous medium
were not available from the literature, it can be assumed that
electron transfer is favored and exciplex formation is disfavored
in comparison with acetonitrile.
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ARTICLE
Figure 6. Relationship between site I (blue) and site II (black) contribution in the presence of (A) oleic acid and (B) ibuprofen.
component was associated with the presence of free 2 in the bulk
solution, already determined in PBS (see above). However, the
other two longer lifetimes can be correlated with the existence of
two HSA-bound species.
’ ACKNOWLEDGMENT
Financial support from the MICINN (grants: CTQ2010-
99010) and the Generalitat Valenciana (Prometeo Program) is
gratefully acknowledged.
To support further this assignment, LFP experiments were
performed in the presence of oleic acid, a well-established
displacing probe for site I ligands.32 The addition of this probe
resulted in a reduced contribution of the longest lifetime
component. Thus, at 1:2.5:2.5 2/HSA/oleic acid molar ratio,
this value dropped to ca. 23% (Figure 5), as compared with 50%
in the absence of oleic acid.
Thus, the longest lifetime (14.4 μs) component is confirmed
as corresponding to 2 within site I, whereas the 1.4 μs component
can be safely assigned to site II-bound 2. Further support for this
assignment was obtained upon the addition of ibuprofen, a site II
displacer.33 As expected, the results (Figure 6B) showed the
opposite trend to that shown by oleic acid, revealing a diminished
contribution of 2 in site II with increasing amounts of ibuprofen,
accompanied by a slight increase in 2 in site I.
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’ CONCLUSIONS
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Using the 4-methoxy and 4,4-dimethoxy derivatives of benzo-
phenone (1, 2), the two types of triplets (nπ*and ππ*) can be
simultaneously detected in the same medium by transient absorp-
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dent on the polarity of the solvent; the ππ* band (430ꢀ450 nm) is
predominating in aqueous medium, whereas the nπ* absorption
(525ꢀ550 nm) is enhanced in nonpolar solvents.
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’ AUTHOR INFORMATION
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Corresponding Author
*Fax: +34 963877809. E-mail: mmiranda@qim.upv.es (M.A.M.).
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