Solvochromic effects in model eumelanin compounds

Article abstract of DOI:10.1111/j.1751-1097.2007.00290.x

We have created an indolic compound which is ideally suited to the study of the relationship between structure and function in eumelanin formation. N-methyl-5-hydroxy-6-methoxyindole (MHMI) is stable in solid and liquid states, highly soluble in a variety of solvents and forms a dimer only through the 4-4′ positions. The limited binding possibilities are due to functional groups strategically placed to inhibit chemical interactions through the 2 and 7 positions. It forms a crystal structure with a remarkable packing arrangement, with four monomers grouped in parallel pairs spaced 3.5 A apart within each unit cell. Optical spectra reveal a multi-peaked absorbance profile similar to 5,6-dihydroxyindole (DHI) and N-acetyl-tryptophanamide (NATA), and strong fluorescence emission with radiative quantum yields of 29% and 33% in benzene and acetonitrile, respectively. The quantum yield is similar to that of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and shows that solvent aromaticity by itself does not affect the yield. Solution in chloroform results in an almost complete quenching of the fluorescence but an increase in emission is observed with photoactivation. Crystallographic results shown here suggest new structural possibilities for eumelanin and the controlled binding possibilities make this an excellent model for monitoring changes in function with increasing oligomer size in eumelanin formation.

Full text of DOI:10.1111/j.1751-1097.2007.00290.x

Photochemistry and Photobiology, 2008, 84: 620–626
Solvochromic Effects in Model Eumelanin Compounds†
Stephen P. Nighswander-Rempel*1, Indumathy B. Mahadevan1, Paul V. Bernhardt2,
Jessica Butcher1 and Paul Meredith1
1Centre for Organic Photonics and Electronics, School of Physical Sciences,
University of Queensland, Brisbane, Queensland, Australia
2School of Molecular and Microbial Sciences, University of Queensland,
Brisbane, Queensland, Australia
Received 30 October 2007, accepted 8 December 2007, DOI: 10.1111 ⁄ j.1751-1097.2007.00290.x
similar to those of eumelanin and similarly attributed to
structural disorder (7).
ABSTRACT
We have created an indolic compound which is ideally suited to
the study of the relationship between structure and function in
eumelanin formation. N-methyl-5-hydroxy-6-methoxyindole
(MHMI) is stable in solid and liquid states, highly soluble in
a variety of solvents and forms a dimer only through the 4-4¢
positions. The limited binding possibilities are due to functional
groups strategically placed to inhibit chemical interactions
through the 2 and 7 positions. It forms a crystal structure with
Eumelanin is known to consist of macromolecules of 5,6-
dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid
(DHICA) and their oxidized forms. How these units are
arranged within the larger eumelanin complex remains uncer-
tain, and indeed, there are a variety of ways in which
complexes have been suggested to form (8–10). It seems likely
that a variety of macromolecules do form and that this
diversity, or disorder, lies at the heart of eumelanin’s func-
tionality. Indeed, these macromolecules could be responsible
for the low radiative quantum yield, as recent studies have
shown that quantum yield decreases not only with aggregation
(11,12) but also with increasing particle size (13).
In order to understand the relationship between macro-
molecular size and spectroscopic properties more fully, it
would be useful to monitor the spectroscopic properties of one
of these molecules at several levels of polymerization. How-
ever, this is challenging with DHI and DHICA because DHI
tends to react even under mild conditions, making it difficult to
obtain pure monomeric or dimeric samples (12), and DHICA
dimerizes in the 4, 7 and possibly even the 3 position, yielding
several structurally and possibly optically distinct compounds
(14,15). In this report, we present a new compound, N-methyl-
5-hydroxy-6-methoxyindole (MHMI), which is ideally suited
for such studies, and we examine its properties using crystal-
lography, spectroscopy and nuclear magnetic resonance. This
compound has a methyl group attached to the nitrogen in the
pyrrole ring, and a hydroxy group and a methoxy group
attached to the 5 and 6 positions, respectively, in the benzene
ring (Fig. 1). It is an ideal model for studying eumelanin
formation and functionality because the presence of these
functional groups sterically restricts the number of binding
sites, allowing greater control over the possible dimeric and
polymeric species created and greater uniformity than can be
found in natural eumelanin.
a
remarkable packing arrangement, with four monomers
˚
grouped in parallel pairs spaced 3.5 A apart within each unit
cell. Optical spectra reveal a multi-peaked absorbance profile
similar to 5,6-dihydroxyindole (DHI) and N-acetyl-tryptoph-
anamide (NATA), and strong fluorescence emission with
radiative quantum yields of 29% and 33% in benzene and
acetonitrile, respectively. The quantum yield is similar to that
of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and shows
that solvent aromaticity by itself does not affect the yield.
Solution in chloroform results in an almost complete quenching
of the fluorescence but an increase in emission is observed with
photoactivation. Crystallographic results shown here suggest
new structural possibilities for eumelanin and the controlled
binding possibilities make this an excellent model for monitor-
ing changes in function with increasing oligomer size in
eumelanin formation.
INTRODUCTION
Eumelanin is a primary pigment which plays important
photoprotective roles in human skin. It is able to protect us
from the damaging effects of solar UV radiation because of
its broad absorbance profiles, its extremely low quantum
yield and its antioxidant and free radical-scavenging behavior
(1–3). The cause of these highly unusual spectroscopic
properties has been the source of much debate, but recent
work has shown them to result from chemical disorder in the
primary and secondary structures (4–6). This is supported by
recent work on humic substances, whose absorbance, emis-
sion and radiative quantum yield properties are remarkably
MHMI exhibits spectroscopic properties similar to DHICA,
interesting stacking features which shed light on possible
eumelanin structural arrangements, and significant solvochro-
mic behavior. The latter was examined using aromatic
(benzene) and nonaromatic solvents (acetonitrile and chloro-
form), showing that solvent aromaticity by itself has little
effect on radiative quantum yield. Yields for the compound in
chloroform were orders of magnitude lower than in the other
†This invited paper is part of the Symposium-in-Print: Melanins.
*Corresponding author email: snighrem@physics.uq.edu.au (Stephen P.
Nighswander-Rempel)
ꢀ 2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/08
620

Photochemistry and Photobiology, 2008, 84 621
Figure 2. ORTEP3 diagram of
a single molecule (A) from the
structure (30% probability ellipsoids are shown). Note that numeric
position labeling used by ORTEP is different from that found in Fig. 1
and used roufinely in melanin literature, particularly with respect to
positions 2 and 3.
Optical spectroscopy. Absorbance spectra were recorded using a
Cary (Palo Alto, CA) 300 spectrophotometer with a 600 nm min)1
scan speed and 2 nm spectral bandwidth. All spectra were collected
using a 1 cm square quartz cuvette. Solvent scans (obtained under
identical conditions) were used for background correction. Fluores-
cence emission spectra were recorded using a Jobin Yvon (Edison, NJ)
Fluoromax 3 fluorimeter with a 2 nm bandpass, an integration time of
0.5 s and an excitation wavelength of 300 nm. Solvent scans were
again performed under identical instrumental conditions for back-
ground correction. All emission spectra were corrected for reabsorp-
tion and inner filter effects using the method outlined previously (3).
Quantum yields were calculated using the method described previously
(20) and quinine sulfate as a reference standard (21). As the quantum
yield of quinine is temperature-dependent, the ambient temperature
surrounding the cuvette was measured to be 35ꢁC, resulting in a 2.5%
shift from the published value of 0.546.
Figure 1. Structures of indolic compounds. Numeric labeling of the
indole positions is included for discussion in the text.
solvents and we examine the chemical interactions responsible
for that. Results for dimeric and oligomeric forms of MHMI
will be published separately.
MATERIALS AND METHODS
Compound synthesis. 5-Benzyloxy-6-methoxyindole purchased from
Sigma Aldrich was N-methylated in dry DMF using sodium hydride as
the base and methyl iodide as the alkylating agent. The reaction was
carried out under an atmosphere of argon for 2 h. The N-methyl-5-
benzyloxy-6-methoxyindole (MBMI) thus obtained was subjected to
hydrogenolysis for 3 h in ethyl acetate–acetic acid solution, using 5%
palladium on carbon as catalyst to furnish MHMI monomer. The
crude product thus obtained was purified over a column of silica using
chloroform as eluant, and then crystallized from dichloromethane-
hexane. A series of solutions ranging in concentration from 0.5 to
10 lM were prepared in each of benzene, acetonitrile and chloroform.
Crystallography. Intensity data at 293 K were collected on an
Enraf-Nonius CAD4 four-circle diffractometer using graphite mono-
NMR spectroscopy. 1H NMR and 13C NMR spectra were recorded
on a Bruker Digital Avance-500 NMR spectrometer, in d4-methanol.
The chemical shifts are reported in p.p.m. relative to d4-methanol,
3.30 p.p.m. for proton and 49.0 p.p.m. for 13C.
RESULTS
The crystal structure of MHMI was determined by X-ray
diffraction. Curiously, the compound crystallizes with four
distinct MHMI monomers in the asymmetric unit. Each
molecule (A, B, C and D) exhibits the same conformation
and interatomic dimensions within experimental uncertainty
and a view of one of these (molecule A) is shown in Fig. 2.
There is an acute intramolecular H-bond between the hydroxyl
and methoxyl groups. The packing is remarkable (Fig. 3a) and
shows that the four distinct molecules are organized into
parallel pairs offset from one another (Fig. 3b). The interpla-
˚
chromated Mo-Ka radiation (k = 0.71073 A) in the x-2h scan mode.
Lattice dimensions were determined by a least squares fit of the setting
parameters of 25 independent reflections. Data reduction was per-
formed with the WINGX package (16). The structure was solved by
direct methods with SHELXS and refined by full matrix least squares
analysis with SHELXL97 (17). All non-H atoms were refined with
anisotropic thermal parameters, and H-atoms were constrained at
estimated positions. The atomic nomenclature is defined in Fig. 2,
drawn with ORTEP-3 (18) while the packing diagrams (Fig. 3) were
drawn with PLATON (19). CCDC 657032 contains the supplementary
crystallographic data for this paper in CIF format. These data can be
obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.
html.
˚
nar separations between the pairs span the range 3.46–3.55 A.
˚
This is nearly identical to the 3.45 A spacing previously
inferred from X-ray scattering studies and applied to a stacking
model in amorphous eumelanin (22). It can be seen that each
monomer is related by a pseudo (noncrystallographic) two-fold
rotation axis to its partner within each pairing (Fig. 3a;
pairings of molecules A ⁄ B and molecules C ⁄ D). These dimers
are oriented with their planes approximately parallel with the
crystallographic a axis (Fig. 3b).
MHMI: C10H11O2, M 177.20, T 293 K, monoclinic, space group
˚
˚
P21 ⁄ a (No. 14, variant of P21 ⁄ c), a 13.928(2) A, b 10.520(2) A, c
24.782(2) A, b 93.47(1)ꢁ, V 3625(1) A , Dc (Z = 16) 1.299 g cm)3
,
3
˚
˚
F(000) 1504, l(Mo Ka) 0.91 cm)1, 6262 unique data (2hmax = 50ꢁ),
Rint = 0.0476, 1511 with I > 2r(I); R 0.0830 (obs. data), wR2 0.2863
(all data), goodness-of-fit 0.935.

622 Stephen P. Nighswander-Rempel et al.
Figure 3. (a) The asymmetric unit of the crystal structure comprising four N-methyl-5-hydroxy-6-methoxyindole (MHMI) molecules organized
into pairs (H atoms omitted for clarity). (b) PLATON diagram showing packing of the MHMI molecules parallel with (a).
Optical absorbance and emission spectra for MHMI are
shown in Fig. 4. The absorbance spectra are interesting in that
they show two distinct peaks and a secondary shoulder. These
features correspond to the energies 4.44 eV (279 nm), 4.15 eV
(299 nm) and 3.99 eV (311 nm). The presence of two or more
distinguishable peaks makes it similar to other indolic com-
pounds, including DHI, N-acetyl-tryptophanamide (NATA)
and indole-3-acetic acid (23–25). These peaks result from two
distinct p-p* transitions with overlapping vibronic bands (24).
Although the absorbance spectrum of NATA has been shown
to be slightly solvent dependent, no such dependence was
observed in MHMI, suggesting that the electronic structure is
not substantially affected by solvent interactions. In particular,
they are virtually identical in acetonitrile and chloroform. In
benzene, which is nonpolar, there is more apparent fine
structure and a slight redshifting, particularly of the shoulder
which now appears as a separate peak. It is also partially
obscured by the strong absorbance of benzene as far as 280 nm.
The emission peaks are also similar in form, and the
Figure 4. Absorbance and emission spectra for N-methyl-5-hydroxy-
emission intensities (relative to the absorbance) are compara-
6-methoxyindole dissolved in benzene (solid line), acetonitrile (dashed
ble in benzene and acetonitrile with quantum yields of 29%
and 33%, respectively. This is also comparable to that of the
line) and chloroform (dotted line). A = 279 nm (4.44 eV),
B = 299 nm (4.15 eV), C = 311 nm (3.99 eV).

Photochemistry and Photobiology, 2008, 84 623
Figure 5. (a) Emission spectra of several N-methyl-5-hydroxy-6-methoxyindole concentrations in chloroform. (b) Plotting integrated emission
against absorbance at 300 nm shows that the intensity of the peak centered at 335 nm scales with concentration. (c) Repeated emission spectra
show that the intensity of the peak centered at 445 nm increases with UV exposure. (d) Integrated emission intensity increases approximately
uniformly with each exposure. The emission intensity does not increase during intermediate time intervals without UV exposure.
DHICA monomer in DMSO (11). When dissolved in chloro-
form, however, the quantum yield is reduced to less than 1%.
Closer inspection of the emission profiles in chloroform reveals
two broad peaks, in contrast to the single peak observed in
benzene and acetonitrile (Fig. 5a). Furthermore, the relative
intensities of the peaks are not constant with either time or
concentration. The short-wavelength peak intensity is linearly
dependent on concentration (Fig. 5b), but the long wavelength
peak is not.
In order to explore this further, we acquired a series of
emission spectra on one sample, exciting at 300 nm. Increased
exposure time to UV light had no effect on the short-
wavelength peak, but caused a dramatic increase in intensity of
the longer wavelength peak (Fig. 5c). It also turned the
solution deep red and this was confirmed by the appearance of
a broad peak centered at 530 nm in the absorbance spectrum.
Time delays between UV exposures caused no increase in peak
intensity, demonstrating that the transformation is photoacti-
vated (Fig 5d). After a sufficient period of exposure, the peak
intensity reached an asymptotic limit, suggesting that the
chemical interactions had reached an equilibrium point. To
test whether this increase in emission was a consequence of
phenol oxidation, the experiment was repeated under a
nitrogen atmosphere. Identical results were obtained, demon-
strating that this emission is not caused by phenol oxidation.
spectroscopic properties) of indolic compounds. This com-
pound is stable in solution, and highly soluble in a variety of
solvents including DMSO and methanol in addition to those
used here. It also crystallizes easily and forms only one form of
dimer (through the 4-4¢ positions), allowing clearer correlation
of structure with spectroscopy. The methyl group attached to
the nitrogen in the pyrrole ring forms a stable bond, inhibiting
binding at the 2 position. Similarly, the methoxy group
sterically hinders binding at the 7 position. Although recent
work has demonstrated the existence of an indolic tetramer
involving bonding in the 3 position (10), this position is still
widely regarded not to contribute significantly to indolic
polymerization and melanin formation in vivo. Absorbance
spectra of the dimer show only one single discernible band,
demonstrating that dimerization results in more closely spaced
vibrational bands (Fig. 6). Proton NMR and crystallography
results demonstrate that covalently coupled MHMI dimers
synthesized in our laboratory were in fact connected in the 4
positions, and no interaction in the 3 position was observed
(Fig. 7). More complete dimer data will be reported elsewhere.
Stable MHMI polymers have also been formed, and these
show a broad absorbance profile similar to that of eumelanin
(Fig. 6). Due to the lack of diversity in the polymer constit-
uents, however, the characteristic peak at 300 nm is still easily
resolved. Preliminary studies of its ultrafast emission dynamics
have already been reported (26).
Crystallographic data for the monomer confirm the molec-
ular structure depicted in Fig. 1 and show that in the solid state,
the two monomers facing each other are rotated 180ꢁ relative to
each other around the long axis and slightly offset (Fig. 3b).
The fact that the monomer forms this pairing arrangement, and
DISCUSSION
We believe that the MHMI monomer created in our labora-
tory and studied here is an ideal compound for studying the
impact of oligomeric size on the functional characteristics (e.g.

624 Stephen P. Nighswander-Rempel et al.
benzene and acetonitrile. Thus, solvent aromaticity by itself
does not affect the MHMI quantum yield. The comparable
emission of MHMI to that of DHICA also demonstrates that
addition of the methyl and methoxy groups in the 1 and 6
positions has little effect on quantum yields.
The reduced emission in chloroform is not caused by
stacking, but the presence of two emission peaks suggested
photoactivated creation of an excimer. Previous studies of
NATA with chloroform demonstrated a strong chemical
reaction between the excited state indole side chain and
chloroform, resulting in a decrease in indole fluorescence and
an increase in fluorescence at 480 nm, similar to that observed
in Fig. 5c (27). The decrease in indole fluorescence seen here is
very small but present, with an isosbestic point appearing at
360 nm. The reduced loss of emission intensity might be due to
the presence of the hydroxy and methoxy groups. In Edwards
et al. (27), appearance of the long-wavelength emission band
was attributed to the binding of a chloroform derivative to the
5 position in indole.
However, the interaction observed here differs from that
reported for NATA in two significant respects. First, as
already noted, the decrease in indole fluorescence with
irradiation is far less substantial than that for NATA. Second,
1H NMR spectra of MHMI in d4 methanol acquired before
and after 2 h of UV irradiation (254 nm) in chloroform were
nearly identical, except for the complete disappearance of the
H-3 doublet and the collapse of the H-2 doublet into a singlet
(data not shown). 13C NMR spectra of the compound before
Figure 6. Absorbance spectra of the N-methyl-5-hydroxy-6-methoxy-
indole dimer (solid) and polymer (dashed) in acetonitrile.
that the spacing is nearly identical to the spacing inferred from
X-ray crystallographic studies of eumelanin, suggests that
perhaps this arrangement is present in eumelanin as well. The
fact that the monomer’s radiative quantum yield in the liquid
phase was substantial and comparable to that of DHICA
indicates that pi-stacking was also negligible in solution in both
Figure 7. Proton NMR spectrum of the N-methyl-5-hydroxy-6-methoxyindole dimer in d4 methanol. Signals at 5.8, 6.9 and 7.0 p.p.m. represent
hydrogen atoms bound to the 3, 2 and 7 positions, respectively. Absence of the H-4 signal indicates that the dimer is bound only in the 4-4¢ position.

Photochemistry and Photobiology, 2008, 84 625
Figure 8. 13C NMR spectra of the N-methyl-5-hydroxy-6-methoxyindole monomer in d4 methanol in its pure form, after deuteration of the 3
position, and after irradiation in chloroform. Irradiation results in deuteration of the 3 position, causing a splitting of the peak at 100 p.p.m. into a
triplet.
and after irradiation also show no change in signal positions
except for the disappearance of the C-3 singlet at 100 p.p.m.
and the appearance of a triplet at 100 p.p.m. because of
deuterium exchange in d4 methanol in the 3 position (Fig. 8).
This deuterium exchange also explains the absence of the
H-3 proton and the H-2 singlet in the irradiated species. If
a chlorine derivative had bound to the monomer upon
irradiation as observed for NATA, there would have either
been a significant signal shift due to the electronegativity of
chlorine or the appearance of an additional signal due to the
presence of another carbon. The observation of neither suggests
that there is no binding between the excited state indole and a
chloroform derivative in this case, but rather that MHMI is
more robust than other indolic compounds. In this case, it is
possible that the negligible quantum yield in chloroform is the
result of interactions such as hydrogen bonding between the
indole and the solvent or excimer formation.
CONCLUSIONS
The MHMI monomer introduced here is well suited to the
study of structure–function relationships in eumelanin, being
stable in solid and liquid states, highly soluble in a variety of
solvents and able to polymerize in a controlled, stepwise
manner. Crystallographic examination reveals a unique struc-
ture in which four monomers are arranged in parallel pairs,
˚
spaced 3.5 A apart, within each unit cell of the crystal. Optical
spectra show it as having a multi-peaked absorbance profile
and overlapping absorbance and emission spectra. More
significantly, it exhibits strong fluorescence emission in solu-

626 Stephen P. Nighswander-Rempel et al.
11. Olsen, S., J. Riesz, I. Mahadevan, A. Coutts, J. P. Bothma, B. J.
Powell, R. H. McKenzie, S. C. Smith and P. Meredith (2007)
Convergent proton-transfer photocycles violate mirror-image
symmetry in a key melanin monomer. J. Am. Chem. Soc. 129,
6672–6673.
tion with quantum yields of 29% and 33% in benzene and
acetonitrile, respectively, similar to that of DHICA. However,
like other indolic compounds, fluorescence is almost com-
pletely quenched in chloroform.
12. Tran, M. L., B. J. Powell and P. Meredith (2006) Chemical and
structural disorder in eumelanins: A possible explanation for
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13. Nofsinger, J. B. and J. D. Simon (2001) Radiative relaxation of
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14. Pezzella, A., A. Napolitano, M. d’Ischia and G. Prota (1996)
Oxidative polymerisation of 5,6-dihydroxyindole-2-carboxylic
acid to melanin: A new insight. Tetrahedron 52, 7913–7920.
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Acknowledgements—The authors thank Dr. Seth Olsen, Prof. Paul
Burn and Prof. Halina Rubinsztein-Dunlop for helpful discussions,
and Dr. Lynette Lambert and Mr. Ben Langley for help with the NMR
and sample preparation. S.P.N.-R. was funded by a post-doctoral
fellowship from Canada’s Natural Sciences and Engineering Research
Council and P.M. was funded by a Smart State Senior Fellowship from
the Queensland State Government (Dept. of State Development).
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