A blocked diketo form of avobenzone: Photostability, photosensitizing properties and triplet quenching by a triazine-derived UVB-filter

Article abstract of DOI:10.1111/j.1751-1097.2008.00414.x

Novel sunscreens are required providing active protection in the UVA and UVB regions. On the other hand, there is an increasing concern about the photosafety of UV filters, as some of them are not sufficiently photostable. Avobenzone is one of the most fr

Full text of DOI:10.1111/j.1751-1097.2008.00414.x

Photochemistry and Photobiology, 2009, 85: 178–184
A Blocked Diketo Form of Avobenzone: Photostability, Photosensitizing
Properties and Triplet Quenching by a Triazine-derived UVB-filter
1
Cecilia Paris , Virginie Lhiaubet-Vallet , Oscar Jimenez , Carles Trullas and Miguel Angel Miranda*
1
2
2
1
´
´
1
´
Instituto de Tecnologıa Quımica UPV-CSIC, Universidad Politecnica de Valencia, Valencia, Spain
´
´
2ISDIN S.A., Dermopharmaceutical Discovery Unit, Barcelona, Spain
Received 25 April 2008, accepted 7 June 2008, DOI: 10.1111/j.1751-1097.2008.00414.x
By contrast with the wide range of UVB filters, appropriate
UVA filters are rare; among them 4-tert-butyl-4¢-meth-
oxydibenzoylmethane (BM-DBM, also known as avobenzone,
Parsol 1789, etc.) is probably the most important representa-
tive. This active ingredient is present in numerous commercial
sunscreen and cosmetic formulations. Avobenzone shows a
strong absorption with maximum at ca 340–350 nm, which
decreases under UV irradiation, resulting in a loss of the UVA
protecting effect (1,2).
The photophysics of BM-DBM has been described and
associated with that of dibenzoylmethanes. It is well
documented that these derivatives lose much of their UVA
protective capability after irradiation through tautomeriza-
tion, fragmentation and photoproduct formation. Indeed,
avobenzone can exist in solution in different tautomeric
forms. In the ground state, it exhibits a keto–enol equilib-
rium where the ‘‘chelated’’ enol form is favored. After UV
light absorption, extensive photoisomerization to the b-
diketone occurs (absorption at 260–280 nm); in the dark, the
enol form is slowly recovered (3). Irreversible photodegra-
dation of BM-DBM is observed, due to a Norrish Type-I
process occurring from the diketone triplet state (3,4).
Avobenzone photostability has been shown to be very
sensitive to the properties of the solvent environment, being
relatively stable in polar protic solvents and markedly
photolabile in nonpolar media (4,5). However, the photo-
chemistry of avobenzone is complex and difficult to under-
stand, due to the presence of tautomers that exhibit different
properties.
ABSTRACT
Novel sunscreens are required providing active protection in the
UVA and UVB regions. On the other hand, there is an increasing
concern about the photosafety of UV filters, as some of them are
not sufficiently photostable. Avobenzone is one of the most
frequently employed sunscreen ingredients, but it has been
reported to partially decompose after irradiation. In the present
work, photophysical and photochemical studies on a methylated
avobenzone-derivative have shown that the diketo form is
responsible for photodegradation. A transient absorption was
observed at 380 nm after laser flash photolysis excitation at
308 nm. It was assigned to the triplet excited state of the diketo
form, as inferred from quenching by oxygen and b-carotene. This
transient also interacted with key building blocks of biomolecules
by triplet–triplet energy transfer (in the case of thymidine) or
electron transfer processes (for 2¢-deoxyguanosine, tryptophan
and tyrosine). Irradiation of the avobenzone derivative in the
presence of a triazine UV-B filter (E-35852) diminished the
undesirable effects of the compound by an efficient quenching of
the triplet excited state. Thus, sunscreen formulations including
triplet quenchers could provide effective protection from the
potential phototoxic and photoallergic effects derived from poor
photostability of avobenzone.
INTRODUCTION
There is an increasing need for safe and efficient topical
sunscreens to prevent harmful effects of UV radiation, such as
cutaneous photoaging and photocarcinogenesis, on human
skin.
Photostability is one of the most important properties of an
effective sunscreen. However, most of the compounds used as
UV-filters exhibit some photoreactivity leading to the forma-
tion of photoproducts that can absorb in different spectral
regions, thus reducing their photoprotective efficacy. More-
over, UV filters can also display some photosensitizing effects:
their photochemical intermediates or stable photoproducts can
interact with skin components, mediating phototoxic and ⁄ or
photoallergic processes. In addition, as new compounds of
unknown toxicology can be formed, evaluation of potential
hazards associated with the use of a sunscreen requires a
detailed study of its photochemistry.
Scheme 1.
Laser flash photolysis of BM-DBM has shown a long-lived
transient at 300 nm, assigned to the nonchelated enol
(Scheme 1), together with a negative band (bleaching) at
360 nm corresponding to the disappearance of the starting
compound (5,6). A previous study of preirradiated solutions of
´
*Corresponding author email: mmiranda@qim.upv.es (Miguel Angel Miranda)
ꢀ 2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/09
178

Photochemistry and Photobiology, 2009, 85 179
chromatography employing a mixture of hexane–ethyl acetate 60:40
v ⁄ v as eluent. 1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)-2-methyl-
propane-1,3-dione (BM-DBM-Me) was isolated with 67% yield and
characterized by NMR and mass spectrometry. 1H NMR (300 MHz,
CD3CN): d = 7.98 (d, J = 8.67 Hz, 2H), 7.92 (d, J = 8.85 Hz, 2H),
7.54 (d, J = 8.67 Hz, 2H), 7.02 (d, J = 8.85 Hz, 2H), 5.46
(q, J = 6.97 Hz, 1H) 3.87 (s, J = Hz, 3H), 1.47 (d, J = 6.97 Hz,
3H), 1.33 (s, 9H). 13C NMR (75 MHz, CD3CN): d = 196.5 (CO),
196.2 (CO), 163.6 (C), 156.9 (C), 133.0 (C), 130.5 (CH), 127.9 (CH),
125.6 (CH), 113.8 (CH), 55.1 (CH3), 49.5 (C), 34.5 (C), 29.9 (CH3),
13.4 (CH3). HRMS (EI) FAB-HRMS m ⁄ z: calcd. for C21H25O3
(MH+) 325.1804; found: 325.1808.
BM-DBM has reported detection of the diketo form triplet
state (7); however, yet, no studies have been performed on the
pure diketo form of dibenzoylmethane derivatives. Its triplet
state or the Norrish Type-I radicals derived therefrom can be
potentially reactive towards skin components or biological
substrates. Indeed, the photosensitizing ability of BM-DBM
towards biologically relevant targets has been evidenced
through strand breaks formation of plasmid DNA (8),
oxidative modification of albumin (9), decreased survival of
yeast cells (10), cytotoxicity to human keratinocytes (11) and
lipid peroxidation (12).
Thus, stabilizers have been incorporated within the sun-
screen formulations like cinnamates, benzylidene malonates,
benzylidene camphor or triazines (7,13).
In the present study, we have investigated the photo-
physical caracteristics of a blocked diketo form derived from
BM-DBM, then we have examined its photoreactivity
towards key biomolecule building blocks, and finally we
have evaluated the potentially protective effect of a new
triaryl-substituted triazine with UVB broadband filtering
ability (Chart 1).
UV-vis absorption spectroscopy. Absorbances of the samples were
recorded with a double beam Varian UV-vis-NIR model Cary5G
spectrophotometer, using 1 cm pathway quartz cuvettes.
Time-resolved absorption spectroscopy. The laser photolysis system
employed for the studies was based on a pulsed XeCl Excimer Laser
(LEXTRA50 Lambda Physik LaserTechnik), which used a 308 nm as
excitation wavelength. The single pulses for excitation of the samples
were of about 40 mJ per pulse, 10 ns per pulse. A Lo255 Oriel Xenon
lamp was employed as a detecting light source. The laser flash
photolysis apparatus consisted of a pulsed laser, a Xe lamp, a
monochromator, a photomultiplier (PMT) system and an oscilloscope.
The output signal from the oscilloscope was transferred to a personal
computer.
All the spectroscopic measurements were performed at room
temperature, adjusting the 308 nm solution absorbances at 0.3–0.35.
If not specified, the samples were purged with N2 before each
measurement.
Oxygen quenching rate constants were determined from air- and
oxygen-purged acetonitrile solutions of BM-DBM-Me and taking into
account oxygen concentration of 1.9 · 103 M and 9.1 · 103 M for air
and oxygen saturated solutions, respectively (14). For the rest of the
quenching experiments, stock solutions were prepared in water for
dGuo, thymidine (Thd), Tyr and Trp, in DMSO for E-35852 and in
benzene for b-carotene.
Singlet oxygen measurements. Singlet oxygen formation was
observed by monitoring the phosphorescence at 1270 nm upon laser
excitation of an optically matched solution (A = 0.3) of BM-DBM-
Me relative to a standard solution of perinaphthenone, which exhibits
a /D value of 1.0 in acetonitrile. The quantum yields for singlet oxygen
formation were determined from the slope of the plots of signal
intensity at zero time versus laser light intensity, employing Eq. (1), and
using acetonitrile as solvent in all cases. A set of neutral density filters
was employed to obtain different laser intensities.
Isample
/DðsampleÞ ¼
/DðperinaphthenoneÞ
ð1Þ
Chart 1.
Iperinaphthenone
where Isample is the emission intensity for the sample, Iperinaphthenone is
the emission intensity for perinaphthenone (standard) and /D(peri-
naphthenone) is the quantum yield of singlet oxygen formation for
perinaphthenone.
MATERIALS AND METHODS
4-tert-butyl-4¢-methoxydibenzoylmethane (BM-DBM, avobenzone)
>99% purity was purchased from Fluka and used as received. 2-(1-
Benzyl-1H-pyrrol-2-yl)-4,6-bis[4-(2-ethylhexyloxy)-2-hydroxyphenyl]-
1,3,5-triazine (E-35852) was kindly provided by ISDIN S.A. (Barce-
lona, Spain).
Iodomethane, L-tyrosine, L-tryptophan, 2¢-deoxyguanosine (dGuo),
thymidine and perinaphthenone, as well as all the solvents, were
purchased from Sigma-Aldrich and used without further purification.
Toluene, benzene and dimethyl sulfoxide (DMSO) were of spectros-
copy grade, whereas acetonitrile, acetone and methanol were of HPLC
grade. Sodium phosphate aqueous buffer solution (pH = 7) was used
to dissolve the amino acids.
The luminescence (1270 nm) from singlet oxygen was detected by
means of an Oriel 71614 germanium photodiode (5 mm2) coupled to
the laser photolysis cell in right-angle geometry. An excimer laser
operated with a Xe ⁄ HCl gas mixture (LEXTRA50 Lambda Physik
LaserTechnik) was used for the excitation (k = 308 nm, 30 mJ per
pulse, 10 ns per pulse). A 5 mm thick (5 cm diameter) 1050 nm
cut-off silicon filter and a 1270 nm interference filter were placed
between the diode and the cell. The photodiode output current was
amplified and fed into a TDS-640A Tektronix oscilloscope via a
Co-linear 150 MHz, 20 dB amplifier. The output signal from the
oscilloscope was transferred to a personal computer for study.
Steady-state photolysis of BM-DBM-Me. Photolysis of 5 · 10)4
M
Preparation
of
1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)-2-
acetonitrile solution of BM-DBM-Me in the presence and absence of
E-35852 was performed to evaluate the photoprotection properties
of the latter compound. Broadband irradiations of the solutions
were carried out using an OrielClass A91192A solar simulator
(Stratford, CT) with a 1000 W Xe arc. Its output was adequately
methylpropane-1,3-dione. Avobenzone (1 g, 3.2 mmol) was dissolved
in 50 mL of acetone and reacted overnight with an excess of
iodomethane (1.9 g, 12.9 mmol) and KCO3 (0.9 g, 6.4 mmol) under
a nitrogen atmosphere at 40–50ꢁC. Then, the crude was filtered under
vacuum, to separate the KI formed as a white powder, reserving the
organic extract. After the removal of the solvent under reduced
pressure, the viscous material was purified by silica gel column
filtered to produce
(1.5 G air mass filter). The spectral output was measured as ca
1000 W m)2
a spectrum approximating natural sunlight
.

180 Cecilia Paris et al.
Photophysics of methylated avobenzone
RESULTS AND DISCUSSION
The photophysical behavior of BM-DBM-Me (the blocked
diketo form) was studied by means of time-resolved nanosec-
ond laser flash photolysis, performed at 308 nm with a pulsed
excimer Xe ⁄ Cl laser. In acetonitrile, a broad transient absorp-
tion spectrum with maximum at 380 nm was observed (Fig. 2);
the decay showed a first-order kinetics corresponding to a
lifetime of ca 0.85 ls.
The obtained transient was tentatively assigned to the triplet
state (3BM-DBM-Me*) by comparison with the spectrum
reported in the literature for a preirradiated solution of BM-
DBM (absorption in the range 300–500 nm, s ꢀ500 ns) (7).
This assignment was confirmed by quenching experiments. As
As stated above, the photoactive form of avobenzone is the
diketo tautomer. Its excited triplet state has been previously
detected by laser flash photolysis of preirradiated samples (7).
Nevertheless, the results obtained with photomixtures can be
misleading due to the presence of varying amounts of the enol
form and other photoproducts that can act as triplet quench-
ers. Hence, to perform a complete characterization of the
excited species, or to address the problem of interaction with
photostabilizing molecules or biological substrates, it would be
desirable to block the diketo form. One efficient way to achieve
this goal could be alkylation in the a-carbonyl position. Along
this line, Wetz et al. have derivatized avobenzone with a C10
alkyl chain and found that the diketo form is mainly obtained;
however, its irradiation results in efficient generation of the
parent filter by a Norrish II process (15). Hence, we decided to
work with the methylated derivative (Chart 1) as a model
diketo compound so as to avoid the photoreactivity due to the
presence of a hydrogen in c-position. In avobenzone itself the
photobehavior is more complex, as it is dominated by the
possibility of excited state proton transfer. This provides a
rather efficient mechanism of deactivation, a desirable prop-
erty for a sunscreen.
expected for a triplet, it was efficiently quenched by oxygen
)1
with kq = 2.2 · 109
M
· s)1 (Fig. 3). Moreover, experi-
ments in the presence of b-carotene clearly showed the
formation of the corresponding triplet state at k = 510 nm
concomitantly with the decay of the 3BM-DBM-Me* signal
Synthesis and characterization of methylated
avobenzone (BM-DBM-Me)
Avobenzone was methylated by reaction with methylene
iodide in nitrogen-bubbled acetone. The NMR spectra in
different solvents (CD3CN, CD3OD and CDCl3) exhibited a
quartet at d = 5.4 ppm and a doublet at d = 1.5 ppm,
characteristic for the hydrogen and the methyl group of the
diketo tautomer. No signal corresponding to the enol form
was detected (see Materials and Methods section).
As shown in Fig. 1, UV-Vis absorption spectrum of BM-
DBM-Me is fully consistent with the keto chromophore. In
acetonitrile, avobenzone is essentially present as a chelated
enol with maximum at 355 nm, while the methylated derivative
exhibits the keto maximum at 260 nm (15).
Figure 2. 308 nm laser flash photolysis of BM-DBM-Me in a N2-
purged acetonitrile solution. Triplet–triplet absorption spectra with
maximum at 380 nm, at different times after the laser pulse.
Figure 1. UV absorption spectra of an acetonitrile solution of avo-
benzone (enol form, dashed line) and BM-DBM-Me (diketo form,
black line).
Figure 3. Quenching of 3BM-DBM-Me* by oxygen as revealed by the
decays under nitrogen (square), air (open circle) and oxygen (triangle)
after laser flash photolysis at 308 nm. Inset: Stern-Volmer plot.

Photochemistry and Photobiology, 2009, 85 181
(Fig. 4). Thus, the triplet–triplet energy-transfer mechanism is
in the origin of the efficient population of the quencher triplet
state, confirming the triplet nature of the 380 nm transient.
Interaction with avobenzone enol form
Potential interactions between the excited diketo form and the
ground state enol form was investigated by studying the
3
quenching of BM-DBM-Me* by BM-DBM. An efficient rate
Singlet oxygen formation
)1
constant of 6.5 · 109
M
· s)1 was obtained; hence, triplet–
The quantum efficiency of singlet oxygen (1O2) formation was
determined employing time-resolved near-IR emission studies
upon laser excitation at 308 nm and using perinaphthenone as
standard (/D = 1.0) (16). The maximum phosphorescence
intensity (kem = 1270 nm) obtained was measured and plotted
as a function of the laser energy for BM-DBM-Me and
perinaphthenone (Fig. 5). From the slopes of the linear plots
obtained, a quantum yield for singlet oxygen formation (/D) of
0.3 was determined which is in agreement with earlier
expectations (7).
triplet energy transfer can be proposed, which would be in
agreement with the triplet energies described in the literature
for the related diketo and the enol forms of dibenzoyl methane
derivatives at 72 kcal mol)1 · s)1 and 59 kcal · mol)1, respec-
tively (4,17).
Photoreactivity of 1O2 towards the BM-DBM enol was also
investigated as this could be the basis of a self-photosensitized
degradation of avobenzone (18). Singlet oxygen was generated
by perinaphthenone, and the decay of its emission at 1270 nm
was recorded as a function of BM-DBM concentration.
Addition of increasing amounts of BM-DBM resulted in a
shortening of singlet oxygen lifetime. The corresponding
)1
quenching rate constant (kq = 3 · 106
M
· s)1
)
was
obtained by means of the Stern-Volmer plot, confirming that
singlet oxygen generated by the diketo form can react with the
enol tautomer.
Interactions with biological components
To evaluate the photobiological risk of avobenzone diketo
form, BM-DBM-Me photosensitization properties were exam-
ined in the presence of biomolecule building blocks.
Two key nucleosides, namely, dGuo and Thd, were chosen
as targets for BM-DBM-Me photosensitization. In the former
case,
the
observed
)1
quenching
rate
constant
(kq = 4.5 · 108
M
· s)1, Table 1) is associated with a chem-
ical reaction, as suggested by the changes in the transient
absorption spectrum. As shown in Fig. 6, BM-DBM-Me triplet
state gives rise to dGuo radical formation that exhibits a very
weak spectrum with maxima at 315, 380 and 540 nm (19).
Accordingly, the involved mechanism appears to be an electron
transfer from dGuo to the BM-DBM-Me excited triplet state.
The second selected nucleoside was Thd; it was also able
Figure 4. b-carotene triplet excited state formation by triplet–triplet
energy transfer from 3BM-DBM-Me*. Transient absorption spectra
were obtained in acetonitrile, upon 308 nm excitation of 3BM-DBM-
Me* in the presence of 16 · 10)6 M b-carotene.
to act as an efficient quencher of 3BM-DBM-Me* with
kq = 8 · 108
M
· s)1 (see Fig. 7 and Table 1). By contrast
)1
with dGuo, no observable spectral changes were detected in this
case (Fig. 7); nevertheless, quenching can be safely attributed to
triplet–triplet energy transfer in view of the slightly exergonic
nature of the process (DG ca )2 kcal · mol)1).
Table 1. Rate constants for quenching of triplet BM-DBM-Me, in
acetonitrile solution measured by laser flash photolysis upon excitation
at 308 nm.
)1
Quencher
kq (M · s)1
)
Oxygen
b-carotene
Avobenzone
Thd
dGuo
Trp
Tyr
2.2 · 109
1.8 · 1010
6.5 · 109
8 · 108
4.5 · 108
2 · 109
3 · 108
Figure 5. Singlet oxygen phosphorescence emission intensity (moni-
tored at 1270 nm) versus relative laser intensity for oxygen saturated
solutions of perinaphthenone (open circle) and BM-DBM-Me (square)
in acetonitrile.
Thd = thymidine; dGuo = 2¢-deoxyguanosine; Trp = tryptophan;
Tyr = tyrosine.

182 Cecilia Paris et al.
Figure 6. Laser flash photolysis of BM-DBM-Me in the presence of
dGuo (3 · 10)3M). Signal obtained 0.07 ls (black line) and 7.5 ls
(dashed line) after the laser pulse. Inset: dGuo radical signal.
Figure 8. Laser flash photolysis of BM-DBM-Me in the presence of
6 · 10)3 M tyrosine (A) or 1.5 · 10)3 M tryptophan (B) at 0.07 ls
(black line) or 7.5 ls (dashed line) after the laser pulse.
Figure 7. Stern-Volmer plot of 3BM-DBM-Me* quenching by thymi-
dine (Thd).
Tyrosine (Tyr) and tryptophan (Trp) were the two amino
acid candidates selected to study the interaction of 3BM-DBM-
Me* with protein components. In both cases, important kq
formulated with other organic UVB filters, to provide a larger
UVA ⁄ UVB protection. This combination has a double-edged
sword effect: avobenzone photodegradation can be retarded
by the presence of the other filters but, on the other hand, it
can also be accelerated if a photoreaction occurs between the
different components (2,12,13).
In this context, a new broadband UVB-filter (E-35852)
was considered as a potential photostabilizer of avobenzone.
In a first approach, laser flash photolysis was performed to
evaluate the interaction between BM-DBM-Me triplet state
and E-35852. In a control experiment, no transient absorp-
tion spectrum was detected in the case of E-35852 alone.
Remarkably, the decay of 3BM-DBM-Me*, monitored at
380 nm, was significantly shortened upon addition of
)1
values were obtained (2 · 109 and 3 · 108
M
· s)1 for Trp
and Tyr, respectively). Tryptophanyl (kmax 310 and 520 nm)
and tyrosinyl radicals (kmax 385 and 410 nm) were detected as
a result of the quenching process (Fig. 8) (19). An electron
3
transfer mechanism between the amino acid and BM-DBM-
Me* should be at the origin of the quenching processes.
Thus, avobenzone is reactive towards biological targets. The
triplet state of diketo form photosensitizes radical formation in
the case of dGuo, Tyr and Trp by an electron transfer
mechanism. The semi-reduced avobenzone-derived radical is
not clearly detected, probably because of its low molar
absorption coefficient (20). Avobenzone also induces, in the
case of thymidine, the triplet–triplet energy transfer mecha-
nism at the origin of thymidine dimer formation.
E-35852; the bimolecular quenching rate constant obtained
)1
from the Stern Volmer plot was 1.8 · 1010
(Fig. 9).
M
· s)1
Then, steady-state photolysis was performed to compare
the fate of BM-DBM-Me when irradiated with or without
E-35852. The solutions were exposed to simulated sunlight
and the kinetics of photodegradation was followed by
HPLC, monitoring the decrease in the area corresponding
to the BM-DBM-Me peak with irradiation time. As shown
Photostabilization by a triazine
In spite of its limited photostability, avobenzone is one of the
most commonly used UVA-filters due to its broadband
absorption. In sunscreens, the active ingredient is generally

Photochemistry and Photobiology, 2009, 85 183
Thd, Tyr and Trp. new triazine-derived UVB-filter
A
(E-35852) acts as an efficient triplet quencher reducing the
inherent photolability and photosensitizing properties of the
diketo form.
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Figure 10. Solar-simulated photolysis of BM-DBM-Me (5 · 10)4 M)
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in Fig. 10, after 24 h of irradiation only 80% of starting
BM-DBM-Me was still present in solution, while in the
presence of E-35852 no significant decomposition was
observed.
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result of two factors, namely the inner filtering effect of
E-35852 (see inset of Fig. 10) and the established triplet state
quenching of BM-DBM-Me by E-35852. In this way, all
photodegradation pathways (Norrish I process, singlet oxygen
formation, etc.) at the origin of BM-DBM-Me decomposition
are disfavored.
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CONCLUSION
In the present work, a simple derivatization of avobenzone has
been used to block the keto-enol equilibrium, for studying the
behavior of the pure diketo tautomer. The excited triplet state,
characterized by laser flash photolysis has been shown to
photosensitize key building blocks of biomolecules like dGuo,

184 Cecilia Paris et al.
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