Role of solvent, pH, and molecular size in excited-state deactivation of key eumelanin building blocks: Implications for melanin pigment photostability

Article abstract of DOI:10.1021/ja806345q

Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380 ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT). The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic analogies with the corresponding processes in DNA and other biomolecules are recognized.

Full text of DOI:10.1021/ja806345q

Published on Web 11/13/2008
Role of Solvent, pH, and Molecular Size in Excited-State
Deactivation of Key Eumelanin Building Blocks: Implications
for Melanin Pigment Photostability
,
†
‡
‡
§
§
M. Gauden,* A. Pezzella, L. Panzella, M. T. Neves-Petersen, E. Skovsen,
§
|
‡
‡
†
S. B. Petersen, K. M. Mullen, A. Napolitano, M. d’Ischia, and V. Sundstr o¨ m
Department of Chemical Physics, Lund UniVersity, Box 124, S 22100 Lund, Sweden,
Department of Organic Chemistry and Biochemistry, UniVersity of Naples Federico II, Naples,
Italy, Department of Physics and Nanotechnology, Aalborg UniVersity, Denmark, and Biophysics
Group, Department of Physics and Astronomy, Faculty of Sciences, Vrije UniVersiteit, De
Boelelaan 1081, 1081 HV Amsterdam
Received August 11, 2008; E-mail: Magdalena.Gauden@chemphys.lu.se
Abstract: Ultrafast time-resolved fluorescence spectroscopy has been used to investigate the excited-
state dynamics of the basic eumelanin building block 5,6-dihydroxyindole-2-carboxylic acid (DHICA), its
acetylated, methylated, and carboxylic ester derivatives, and two oligomers, a dimer and a trimer in the
O-acetylated forms. The results show that (1) excited-state decays are faster for the trimer relative to the
monomer; (2) for parent DHICA, excited-state lifetimes are much shorter in aqueous acidic medium (380
ps) as compared to organic solvent (acetonitrile, 2.6 ns); and (3) variation of fluorescence spectra and
excited-state dynamics can be understood as a result of excited-state intramolecular proton transfer (ESIPT).
The dependence on the DHICA oligomer size of the excited-state deactivation and its ESIPT mechanism
provides important insight into the photostability and the photoprotective function of eumelanin. Mechanistic
analogies with the corresponding processes in DNA and other biomolecules are recognized.
Introduction
on exposure to oxidizing agents or to UV radiation or even on
standing at neutral physiologic pH. It has been proposed mainly
Melanins are a peculiar group of pigments responsible for
the broad range of colorations of mammalian skin, hair, and
eyes. Production of melanins takes place within epidermal
melanocytes by the tyrosinase-catalyzed oxidation of tyrosine.
In the process of melanogenesis, two types of melanin are
formed: eumelanin (black-brown) and pheomelanin (yellow to
reddish brown). The dark eumelanin is thought to be involved
in skin and eye protection against UV damage, mainly because
of its peculiar broad-band monotonic absorption in the
UV-visible range (∼300-800 nm), a photoinducible free
based on X-ray studies that indole monomers are joined into
oligomeric units that interact noncovalently, perhaps by π-π
6
-9
stacking.
As a result, larger aggregates would be formed,
1
0,11
leading to eumelanin particles.
Atomic force microscopy
investigations of the eumelanin isolated from the ink sac of sepia
officinalis suggested that particle formation is a hierarchical
process in which small units are assembled into hundred-
nanometer structures, which then aggregate to form the mor-
1
2-14
phology of the macroscopic pigment.
However, despite
1
considerable efforts, the structures and photophysics involved
in eumelanin photoprotection remain poorly understood. Studies
radical character, and strong nonradiative relaxation of pho-
toexcited electronic states. Nevertheless, solid experimental
evidence supporting the photoprotective concept is still lacking.
Eumelanin is composed of two main monomeric units, 5,6-
dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic
acid (DHICA) at various levels of oxidation and linked in a
1
5
of the optical properties of eumelanin revealed a marked
particle size-dependence: small particles generate long-lived
(
(
5) Tsukamoto, K.; Palumbo, A.; Dischia, M.; Hearing, V. J.; Prota, G.
Biochem. J. 1992, 286, 491–495.
2
random manner. The role of DHICA as a basic eumelanin
6) Zajac, G. W.; Gallas, J. M.; Cheng, J.; Eisner, M.; Moss, S. C.;
Alvarado-Swaisgood, A. E. Biochim. Biophys. Acta, Gen. Subj. 1994,
3
-5
building block is well established
and is illustrated by the
1
199, 271–278.
rapid conversion of this indole to a dark, eumelanin-like pigment
(
(
7) Cheng, J.; Moss, S. C.; Eisner, M. Pigm. Cell Res. 1994, 7, 263–273.
8) Cheng, J.; Moss, S. C.; Eisner, M.; Zschack, P. Pigm. Cell Res. 1994,
7, 255–262.
†
Lund University.
University of Naples Federico II.
Aalborg University.
Vrije Universiteit.
‡
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P. Biophys. J. 1999, 77, 1135–1142.
§
|
(10) Nofsinger, J. B.; Forest, S. E.; Eibest, L. M.; Gold, K. A.; Simon,
J. D. Pigm. Cell Res. 2000, 13, 179–184.
(
1) Seagle, B.-L. L.; Rezai, K. A.; Gasyna, E. M.; Kobori, Y.; Rezaei,
K. A.; Norris, J. R. J. Am. Chem. Soc. 2005, 127, 11220–11221.
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142.
(
7
52.
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3) Ito, S. Biochim. Biophys. Acta 1986, 883, 155–161.
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Tetrahedron 1997, 53, 8281–8286.
1
7038 9 J. AM. CHEM. SOC. 2008, 130, 17038–17043
10.1021/ja806345q CCC: $40.75  2008 American Chemical Society

Deactivation of Key Eumelanin Building Blocks
A R T I C L E S
reactive intermediates upon absorption of UV light, whereby
they may be involved in UV-A-induced photochemical processes
believed to lead to DNA damage, whereas the large particles
efficiently dispose of UV-A energy through rapid nonradiative
decay processes. This size-dependent photoreactivity has been
suggested to be one of the contributing factors to the observed
variations in skin cancer rates among different skin types.
Steady-state fluorescence experiments have shown that the
emission spectra and fluorescence quantum yield depend on the
excitation wavelengths. Time resolved fluorescence and transient
absorption studies have been performed to elucidate excited-
interest of 5,6-dihydroxyindole photochemistry in relation to
the possible involvement of the circulating metabolites in
25
immediate tanning and in the erythemogenic response of skin
to UV radiation. In humans, the serum and urine concentrations
of 5,6-dihydroxyindoles increase dramatically following expo-
sure to sunlight, UVA (315-400 nm) or UVB (290-315 nm),
as well as during PUVA therapy, suggesting that these indoles
may play important biological roles in the response of skin to
actinic damage.
Herein, we provide the first experimental evidence that
DHICA oligomers have shorter excited-state lifetimes than
their monomeric building blocks, a result of direct relevance
for the photoprotective function of eumelanin. The present
work, in addition, determines the fluorescence spectra and
decay times of DHICA at neutral and acidic pH, as well as
in acetonitrile solvent. We show that the variation of spectra
and excited-state dynamics can be understood as a result of
excited-state intramolecular proton transfer, and we propose
this as the mechanism to achieve the photostability of
eumelanin, in analogy with what has been proposed for DNA
1
5-17
state dynamics and photoproduct formation.
Transient
absorption spectra of eumelanins are difficult to interpret,
presumably reflecting quite complex dynamics where several
1
7
processes occur simultaneously. Time resolved fluorescence
18-20
experiments revealed multiexponential decay of fluorescence,
while time-resolved polarization experiments on Sepia eumela-
nin resulted in an ∼80 ps depolarization time constant that was
1
9
attributed to energy transfer between chromophores. Overall,
these observations suggest the existence of several chromophores
2
1
26-28
within the pigment.
and other biomolecules.
Recently, evidence has been presented for mirror-image rule
violation in spectra of DHICA, and it has been proposed that
this phenomenon is due to convergent adiabatic and nonadiabatic
Results
22
Ionization States. Fluorescence measurements were performed
on the following substrates (Figure 1): DHICA, its O-acetyl
excited-state intramolecular proton transfer (ESIPT) processes.
Specifically, excitation into the S or S states of a catecholate
anion of DHICA, represented by dual bands in the absorption
spectrum, leads to emission from the S state of its proton-
1
2
(
DAICA), methyl ester (DHICA-Me), and O,O-dimethyl methyl
ester (DMICA-Me) derivatives, and the symmetric 4,4′-dimer
DHICA-dimer) and trimer (DHICA-trimer), both as the O-
1
(
transfer conjugate. It was proposed that intramonomer ESIPT
may function as an energy dissipation mechanism in the
pigment. Recent calculations have predicted that hydrogen shifts
may be involved in the photophysics of 5,6-dihydroxyindole
acetyl derivatives. Among the molecules studied, only the parent
DHICA is water-soluble. It possesses three potentially ionizable
functional groups, a carboxylic acid at C-2 and two hydroxyl
2
3
groups at C-5 and C-6. The pK
a
’s for these three groups are
(
DHI).
4
.25, 9.76, and 13.2, respectively, as determined by potentio-
Since eumelanin occurs in ViVo at a high degree of aggrega-
29
metric and spectrophotometric titrations. Thus, in the pH range
accessible to experiment, several ionization states and tautomers
can be populated. At pH 3, the fully protonated state dominates;
at pH ∼ 6, the dominant state has only the carboxylic group in
its deprotonated form (anion); at pH > 10, the dominant state
has both the carboxylate and the C-6 hydroxyl deprotonated
tion, it is very difficult to draw detailed conclusions regarding
the reaction mechanism. Already over 30 years ago there were
proposed several mechanisms in order to explain how the
2
4
photoprotection was achieved by the eumelanin pigment. It
has been speculated that energy dissipation occurs through
15
efficient radiationless processes in large aggregates; however,
no direct evidence has been available. In fact, investigation of
the photophysical and photochemical properties of 5,6-dihy-
droxyindole building blocks remains to-date the most useful
approach toward an understanding of photoprotective mecha-
nisms. Time-resolved fluorescence study of excited-state dy-
namics under different conditions might shed new light on the
mechanisms of eumelanin photoprotection. Added to this is the
(
dianion) (see Figure 1B). At pH values of physiological
relevance, i.e. 7.4, DHICA exists as a mixture of the anion and
dianion, in an approximately 1000:1 ratio, and the anion/
protonated form ratio is ∼200:1.
DHICA derivatives, DAICA, and oligomers (DAICA-dimer
and -trimer) were specifically designed to determine the effects
of functional group and molecular size modification on the
fluorescence properties and excited-state dynamics of DHICA.
Functional group modifications reflected by these compounds
include the following: O-acetylation, which substantially blunts
the electron releasing properties of the OH groups; O-methy-
lation, which also does so in alkaline medium, though to a much
lesser extent, preventing pH-dependent ionization; and carboxyl
group conversion to methyl esters, which increases the electron
withdrawing effects on the indole ring and precludes ionization
in aqueous medium and dimerization equilibria in organic
(
16) Nofsinger, J. B.; Ye, T.; Simon, J. D. J. Phys. Chem. B 2001, 105,
2
864–2866.
(
(
17) Ye, T.; Simon, J. D. J. Phys. Chem. B 2003, 107, 11240–11244.
18) Docchio, F.; Boulton, M.; Cubeddu, R.; Ramponi, R.; Barker, P. D.
Photochem. Photobiol. 1991, 54, 247–253.
(
(
19) Forest, S. E.; Lam, W. C.; Millar, D. P.; Nofsinger, J. B.; Simon,
J. D. J. Phys. Chem. B 2000, 104, 811–814.
20) Teuchner, K.; Freyer, W.; Leupold, D.; Volkmer, A.; Birch, D. J. S.;
Altmeyer, P.; Stucker, M.; Hoffmann, K. Photochem. Photobiol. 1999,
7
0, 146–151.
(
(
21) Meredith, P.; Powell, B. J.; Riesz, J.; Nighswander-Rempel, S. P.;
Pederson, M. R.; Moore, E. G. Soft Matter 2006, 2, 37–44.
(25) Maeda, K.; Hatao, M. J. InVest. Dermatol. 2004, 122, 503–509.
(26) Sobolewski, A. L.; Domcke, W.; Dedonder-Lardeux, C.; Jouvet, C.
Phys. Chem. Chem. Phys. 2002, 4, 1093–1100.
22) Olsen, S.; Riesz, J.; Mahadevan, I.; Coutts, A.; Bothma, J. P.; Powell,
B. J.; McKenzie, R. H.; Smith, S. C.; Meredith, P. J. Am. Chem. Soc.
2
007, 129, 6672–6673.
(27) Abo-Riziq, A.; Grace, L.; Nir, E.; Kabelac, M.; Hobza, P.; de Vries,
M. S. Proc. Natl. Acad. Sci. 2005, 102, 20–23.
(
(
23) Sobolewski, A. L.; Domcke, W. ChemPhysChem 2007, 8, 756–762.
24) Pathak, M. A.; Fitzpatrick, T. B. The role of natural photoprotectiVe
agents in human skin, Sunlight and Man: Normal and Abnormal
Photobiologic Responses; University of Tokyo Press: Toyko, 1974.
(28) Sobolewski, A. L.; Domcke, W.; Hattig, C. Proc. Natl. Acad. Sci.
2005, 102, 17903–17906.
(29) Charkoudian, L. K.; Franz, K. J. Inorg. Chem. 2006, 45, 3657–3664.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 50, 2008 17039

A R T I C L E S
Gauden et al.
Figure 2. Absorption spectra of DHICA and its derivatives (DAICA-d)
and oligomers (DHICA-dimer and DHICA-trimer) normalized at the lower
energy band.
Figure 1. (A) Structures of the studied molecules. (B) Protonation equilibria
of DHICA and dominant forms at various pH values.
solvents. Comparison of data from DAICA and the DHICA-
dimer and -trimer was aimed at gaining insights into the effects
of molecular size and oligomerization on the fluorescence of
DHICA, an issue of special relevance to the photophysical
properties of eumelanin pigments.
Figure 3. Fluorescence kinetic traces of DHICA at pH 3, at pH 7, and in
acetonitrile.
Absorption Steady-State Spectra. The eumelanin optical
absorption spectrum is a broad, structureless curve that is
monotonically decreasing as a function of wavelength. DHICA
and its derivatives and higher oligomers all exhibit the same
qualitative spectral features: two main absorption bands, one at
forms. Additionally, we have examined the pH dependence of
the excited-state lifetime of DHICA. We used 280 nm as an
excitation wavelength, and we monitored the fluorescence in
the 350-510-nm wavelength range. Application of a global
analysis procedure yielded a single decay component for all
molecules. In acetonitrile, DHICA has an excited-state lifetime
of 2.6 ns (3 ns after degassing the sample with nitrogen; thus,
further samples were not routinely degassed prior to measure-
ment). Strikingly, the excited-state lifetime in aqueous buffer
solution is much shorter than that in acetonitrile and is pH-
dependent: at pH 3, DHICA has an excited-state lifetime of
∼
225 nm and another at ∼315 nm (Figure 2). The high-energy
band is narrower than the low-energy band, and both bands
exhibit a more or less pronounced substructure. The relative
intensities and peak positions of the bands vary somewhat
between the different derivatives and oligomers.
Time-Resolved Fluorescence. With time-resolved fluores-
cence, we measure the time evolution of the spontaneous
emission following excitation of the molecule. Although the
fluorescence signal is often weak due to low quantum yield, it
has a great ability to exclusively monitor the excited-state
dynamics and thus provide a less complex and more easily
interpreted picture of the photodynamics than transient absorp-
tion, which monitors all species that happen to absorb the probe
light, i.e. both excited states and ground states. We have
performed time-resolved fluorescence streak camera experiments
in order to examine the excited-state dynamics of DHICA in
its acetylated, methylated, and carboxylic ester derivatives as
well as two oligomers: a dimer and a trimer in the O-acetylated
3
80 ps, which increases to 1.2 ns at pH 7. The pH dependence
of the excited-state lifetime is illustrated in Figure 3. As we
will discuss in more detail below, the 3- to 4-fold shorter
excited-state lifetime for DHICA at pH 3 as compared to pH 7
we assign to excited-state intramolecular proton transfer.
A comparison of the kinetic traces at the maximum emission
for DHICA, DHICA-dimer, and DHICA-trimer demonstrates
a shortening of the excited-state lifetime for higher oligomers
(Figure 4). A possible explanation and implications of this result
are discussed below. Fluorescence lifetimes for all the studied
molecules are summarized in Table 1.
1
7040 J. AM. CHEM. SOC. 9 VOL. 130, NO. 50, 2008

Deactivation of Key Eumelanin Building Blocks
A R T I C L E S
3
0
discovered by Weller and has since then been extensively
3
1
studied. The excited-state proton transfer process is often
facilitated if an intramolecular hydrogen bond exists in the
ground state. The COOH and NH groups of the molecules with
protonated carboxylic groups can be envisaged to be involved
3
2
in an intramolecular hydrogen bond. In fact, it was shown
for molecules related to DHICA, indole-2-carboxylic acid, and
indole-5-carboxylic acid that there is an excited-state equilibrium
between the neutral hydrogen bonded form and the zwitterionic
form, where the carboxylic hydrogen has been transferred to
the nitrogen. In most solvents, a very broad and sometimes dual
band fluorescence was observed, with a red-shifted band due
to the zwitterionic form and a blue-shifted indole-like fluores-
cence due to the neutral form. Acidic conditions with a
protonated COOH group and a developed HN----HOOC hy-
drogen bond were seen to give rise to red-shifted zwitterionic
fluorescence, whereas basic conditions favoring the anionic form
(
and thus no possibility of hydrogen bonding and proton transfer)
Figure 4. Fluorescence kinetic traces of DAICA, DAICA-dimer, and
DAICA-trimer in acetonitrile.
gave weak indole-like blue-shifted fluorescence. These results
are directly relevant and applicable to the molecules studied
here. The molecules with a protonated COOH group all exhibit
very broad fluorescence spectra with more or less pronounced
multiple bands or wings (Figure 5). A change of pH from 3 to
Table 1. Summary of Measured Fluorescence Lifetimes
pH 3 (ns)
pH 7 (ns)
acetonitrile (ns)
DHICA
DAICA
DHICA-Me
DMICA-Me
DHICA-dimer
DHICA-trimer
0.4
1.2
2.6
2.6
2.5
2.6
2.5
1.7
7
in aqueous solution results in a ∼30 nm blue-shift of the
fluorescence spectrum of DHICA, similar to what was observed
for indole-2-carboxylic acid and indole-5-carboxylic acid.
3
2
A
spectral band analysis of the fluorescence spectra of the above-
mentioned molecules (Figure 5 inset) shows that the spectra
can be described by two Gaussian bands centered at ∼390-410
and ∼440 nm. The slight variation of the position of the
component spectra for the different DHICA derivatives can be
understood as a substituent effect.
Examining the decay-associated spectra (DAS) of the studied
molecules (Figure 5) reveals that they depend on the substitution
pattern as well as on solvent and pH. In 0.1 M phosphate buffer
at pH 3, the DAS of DHICA has a maximum at ∼440 nm. At
pH 7, the maximum is blue-shifted by ∼30 nm, and in
acetonitrile, there is a slight further blue-shift to ∼408 nm.
Derivatives of DHICA show characteristic spectral shifts in
acetonitrile but cannot be studied in aqueous solution. The
decay-associated spectrum of DHICA-Me presents a maximum
at ∼425 nm, while for DAICA the maximum is at ∼390 nm;
DMICA-Me shows an emission maximum at ∼410 nm. DAS
of the dimer and trimer of DHICA show further spectral
changes: the DHICA-dimer presents a maximum at ∼405 nm,
whereas the DHICA-trimer presents a maximum at ∼450 nm.
The carboxyl and hydroxyl groups of the molecules offer rich
opportunities for intra- and intermolecular hydrogen bonding
and consequently proton transfer in the excited state. We will
show below that much of the spectral variation described above
can be understood as a result of hydrogen bonding and excited-
state proton transfer equilibria involving the COOH and NH
groups (and possibly also the 5,6-OH groups).
In analogy with the assignments made for indole-2-carboxylic
32
acid and indole-5-carboxylic acid, we propose the presence
of an excited-state equilibrium between hydrogen bonded and
zwitterionic forms to explain the fluorescence properties of the
compounds studied here (except for DHICA-Me and DMICA-
Me) (Figure 6). The zwitterionic form is characterized by red-
shifted fluorescence at ∼440 nm, and the neutral hydrogen
bonded form has fluorescence at 390-410 nm. Excited-state
intramolecular proton transfer is known to be very fast (sub-
3
1
picosecond to picosecond time scale), implying that the
excited-state equilibrium is established very quickly, much faster
than the decay of the two excited species and also much faster
than the time resolution of our experiment. This leads to the
observed single exponential fluorescence decays. With this
model, we can now rationalize the observed fluorescence
behavior under the various experimental conditions used. The
red-shifted short-lived (380 ps) fluorescence in acidic aqueous
solution is due to the zwitterionic form, which is stabilized in
the polar protic environment. At neutral pH, the DHICA is in
its anionic form, having blue-shifted fluorescence (390-410 nm;
1
.2 ns lifetime), and in analogy with the picture obtained for
Discussion
3
2
indole-2-carboxylic acid and indole-5-carboxylic acid, this
fluorescence can be assigned to an indole-like fluorescence.
However, the situation is more complex for DHICA possessing
the two 5,6-OH groups, which also can be involved in
H-bonding and proton transfer. Thus, according to the calcula-
We start the discussion by assigning the DAS fluorescence
spectra obtained from the global analysis (Figure 5). Since all
fluorescence decays are single exponential, these spectra are
identical to conventional steady-state fluorescence spectra. The
variation of fluorescence spectra with pH, solvent, and substitu-
tion pattern offers the first clue to an understanding of the
fluorescence properties. Excited-state intramolecular proton
transfer (ESIPT) is known to lead to effects such as dual
fluorescence and fluorescence quenching. The effect was first
(30) Weller, A. Z. Elektrochem. 1956, 60, 1144–1147.
(
31) Kosower, E. M.; Huppert, D. Annu. ReV. Phys. Chem. 1986, 37, 127–
1
56.
(
32) Bangal, P. R.; Chakravorti, S. J. Phys. Chem. A 1999, 103, 8585–
8594.
J. AM. CHEM. SOC. 9 VOL. 130, NO. 50, 2008 17041

A R T I C L E S
Gauden et al.
processes. In conclusion of this part, we propose that DHICA
molecules and their derivatives studied here undergo excited-
state proton transfer processes, which leads to dual fluorescence
and, in aqueous solution, short excited-state lifetime. In analogy
with previously observed ESIPT processes for similar molecules
3
2
(
indole-2-carboxylic acid and indole-5-carboxylic acid ), we
propose that for the parent DHICA the carboxylic proton and
indole nitrogen are important mediators of the process, but for
the molecules with O-acetylation, O-methylation, and carboxyl
group conversion to methyl esters, other proton transfer reactions
could also be active.
Are the results discussed above of relevance to the photo-
protective function of melanin? In acetonitrile, DHICA has a
relatively long excited-state lifetime of 2.6 ns, but in aqueous
solution, it is drastically shortened to 1.2 ns at pH 7 and only
3
80 ps at pH 3. The excited-state lifetime is also decreased in
going from DHICA to the trimer, indicating that the nonradiative
decay is faster for the higher oligomers than for monomers.
This is the first direct experimental evidence that oligomers of
DHICA have shorter excited-state lifetimes than the correspond-
ing monomeric building blocks, and it is consistent with the
view that, in order for the melanin pigment to work as a
photoprotecting agent, the nonradiative decay rate of oligomers
has to be very fast. The oligomers could unfortunately only be
studied in acetonitrile, owing to their low solubility in aqueous
solution. Therefore, we do not know what is the excited-state
lifetime of an oligomer in an aqueous environment at physi-
ological conditions (pH 7). However, it is not unreasonable to
assume that if the shortening of the excited-state lifetime
observed for dimers and trimers in acetonitrile (Figure 4) also
occurs in an aqueous environment, the expected trimer lifetime
would be subnanosecond, and for larger oligomers, it can be
hypothesized to be in the tens to hundreds of picosecond range.
Such short lifetimes would represent a very efficient conversion
of UV-light energy into heat.
Figure 5. Decay-associated spectra (DAS) of the fluorescence decay that
resulted from a global analysis of streak camera experiments on DHICA,
DAICA, and the oligomers DHICA-dimer and DHICA-trimer. The inset
shows band-shape analysis of the fluorescence spectra of DAICA in
acetonitrile.
2
2
tions of Olsen et al., for the anion there is a ground-state
equilibrium between the form with a deprotonated carboxyl
group and the catecholate anion with a deprotonated hydroxyl
attached to the C-6 atom, with the catecholate having lower
2
2
energy. Calculations including a dielectric environment sug-
gested that carboxylate and cathecolate forms are of similar
ground-state energies. The result of these considerations is that,
upon photoexcitation of DHICA in aqueous solution at neutral
pH, the excited state could be deactivated through a similar
ESIPT process, as discussed above for the protonated species,
or through the proton transfer suggested in ref 22 involving the
two hydroxyl groups.
For DHICA and its derivatives in the acetylated forms, the
observed fluorescence spectrum in acetonitrile is a superposition
of fluorescence from the neutral and zwitterionic forms, as
revealed by the broad spectra with underlying band structure
and wings. The varying relative amplitudes of blue and red
bands suggest different excited-state equilibrium constants for
the different molecules. The two methyl esters (DHICA-Me and
DMICA-Me) lacking a carboxylic proton do not have the HN----
HOOC hydrogen bond and proton transfer, but it is well-known
that carboxylic oxygen can be involved in hydrogen bonding
The difference in the excited-state lifetime between monomers
and higher oligomers suggests that a relaxation pathway depends
on the molecular size and structure. The molecular structure
must have been a factor that played a role in evolution in the
selection of a specific molecule for photostability purposes. DNA
is a prominent example of a molecule with photoprotective
mechanisms that prevent UV radiation damage. Although the
biological functions of DNA and melanin are different, they
both need to be photostable. Efficient excited-state deactivation
is critical to DNA photostability, since a long excited-state
lifetime may promote mutagenic reactions. The very short
excited-state lifetimes of DNA bases have been suggested to
have their origins in conical intersections between various
33
34
and ESIPT reactions with alcoholic or amine protons. Thus,
also the methyl esters could be involved in ESIPT reactions
and dual fluorescence. An example of this kind of ESIPT is
3
5
that proposed for salicylic acid. In this case, the reaction is
driven in the opposite direction, from phenol to the carboxylic
group, resulting in formation of a zwitterionic form. Neverthe-
less, there are a few known examples where the nitrogen can
2
6,36,37
excited-state potential surfaces and the ground state,
leading to ultrafast internal conversion processes. Very efficient
excited-state deactivation, specific to the Watson-Crick (WC)
27
3
2
structure of a GC (guanine-cytosine) base pair, was explained
act as a donor and carbon can be an acceptor. In order to
further establish the nature of the ESIPT in DHICAs, quantum
chemical calculations need to be performed. Future experimental
work on variously substituted DHICAs and DHIs (blocking
various proton transfer possibilities) will hopefully also allow
us to better distinguish the role of the different proton transfer
2
8
by Sobolewski et al. as a barrierless proton transfer process
1
that connects the spectroscopic ππ* excited state with the
electronic ground state via two conical intersections. Similarly,
femtosecond mass spectrometry showed that a model base pair
of 2-aminopyridine of WC geometry has a considerably shorter
excited-state lifetime than the monomer or dimers of other
structure. The results were explained as a result of conical
(
(
(
33) Van Benthem, M. H.; Gillispie, G. D. J. Phys. Chem. 1984, 88, 2954–
960.
34) Fahrni, C. J.; Henary, M. M.; VanDerveer, D. G. J. Phys. Chem. A
002, 106, 7655–7663.
35) Lahmani, F.; ZehnackerRentien, A. J. Phys. Chem. A 1997, 101, 6141–
147.
2
(36) Ismail, N.; Blancafort, L.; Olivucci, M.; Kohler, B.; Robb, M. A. J. Am.
Chem. Soc. 2002, 124, 6818–6819.
2
(37) Merchan, M.; Serrano-Andres, L. J. Am. Chem. Soc. 2003, 125, 8108–
8109.
6
1
7042 J. AM. CHEM. SOC. 9 VOL. 130, NO. 50, 2008

Deactivation of Key Eumelanin Building Blocks
A R T I C L E S
Figure 6. Proposed energy level diagram for an excited-state proton transfer in DHICA. Proposed energy level diagram for an excited-state proton transfer
in DHICA.
1
intersections connecting the locally excited ππ* state and the
were sent through a pulse selector (Spectra-Physics) to reduce the
repetition rate from 80 to 8 MHz. The beam of infrared pulses was
then used to pump a harmonic generator (Spectra-Physics, GWU)
to generate the third harmonic of the fundamental wavelength. The
resulting UV output pulses were approximately 200 fs and centered
at 280 nm. The average power of the beam of UV pulses was ∼1.8
mW at a repetition rate of 8 MHz, corresponding to a pulse energy
of 0.2 nJ. These UV pulses were used to excite the sample solution
kept in a rotating cuvette, and the fluorescence emission was
collected using a one inch diameter 50 mm focal length quartz lens
and then focused onto the input slit (width 100 µm) of a
spectrograph (Oriel, MS257). The grating used in the spectrograph
was blazed at 300 nm and had 300 lines/mm. The output from the
spectrograph was sent onto the streak camera (Optronis, GmbH),
and data were collected with a sweep speed of 0.01 mm/ps, which
corresponds to a time resolution of 250 ps at the used entrance slit
of 100 µm. The streak-camera fluorescence data were analyzed
38
electronic ground state with a charge transfer state. More long-
lived so-called excimer states have also been suggested as
intermediates in the excited-state relaxation pathways of oli-
3
9
gonucleotides, with possible roles in photoprotection.
From the discussion above, we conclude that, in both base
pairs of DNA and oligomers of DHICA, which are building
blocks of melanin, excited-state proton transfer reactions (most
likely giving rise to conical intersections) are essential features
for achieving efficient excited-state deactivation. Efficient
excited-state relaxation through similar processes has also been
2
6
40,41
proposed for amino acids and other organic molecules.
Thus, it appears that Nature is using a general and common
theme to achieve photostability and photoprotection of molecules
with key functions.
45
Materials and Methods
globally in terms of a sum of exponential decays, using a parallel
model yielding species associated spectra (DAS) and corresponding
lifetimes.
4
2
43
Sample Preparation. DHICA,
DHICA-Me,
DHICA-
44
44
dimer, and DHICA-trimer were prepared according to literature
procedures. DAICA was prepared by acetylation of DHICA with
acetic anhydride/pyridine. DMICA-Me was prepared by treating
DHICA with an excess of diazomethane in ethyl ether.
Fluorescence Streak Camera Measurements. The near-infrared
output pulses of a femtosecond laser (Spectra-Physics, Tsunami)
Acknowledgment. This work was supported by funding from
the Swedish Energy Agency (STEM), Wennergrenska Stiftelsen,
the Crafoord Foundation, and the Swedish Research Council (VR)
through an excellence grant to the Lund Laser Centre and LaserLab
Europe Contract No. RII3-CT-2003-506350. L.P. thanks
´
“
L’OREAL Italia Per le Donne e la Scienza” for funding.
(
(
(
(
(
38) Schultz, T.; Samoylova, E.; Radloff, W.; Hertel, I. V.; Sobolewski,
A. L.; Domcke, W. Science 2004, 306, 1765–1768.
Note Added after ASAP Publication. Figure 5, originally
39) Crespo-Hernandez, C. E.; Cohen, B.; Kohler, B. Nature 2005, 436,
published November 13, 2008, was replaced by the author.
Wavelength values relating to the figure were changed in the first
and third paragraphs of the Time-Resolved Fluorescence section
and the first and second paragraphs of the Discussion section. The
corrected version was published November 18, 2008.
1
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