Photochemistry and Photobiology, 2008, 84 633
MATERIALS AND METHODS
General. Nuclear magnetic resonance spectra were measured at
1
13
5
00.13 MHz ( H) and 125.03 MHz ( C) using d
as the solvent and referenced to the residual solvent peak at
.30 p.p.m. or 2.49 p.p.m. vs TMS, respectively. Electronic absorption
spectra were measured using 1 cm pathlength quartz cells with solution
4 6
-MeOD or d -DMSO
3
)
6
concentrations of ca 5 · 10 M in DMSO (spectrophotometric grade).
Emission spectra were recorded for all samples using a fluorimeter.
Emission scans were performed between 370 and 600 nm using an
excitation wavelength of 350 nm. A band pass of 4 nm and an
integration of 0.5 s were used. The emission spectra were corrected for
attenuation of the probe beam and reabsorption of the emission
according to a previously published procedure (14). Background scans
were performed under identical instrumental conditions using the
relevant solvent. Electron-impact mass spectral analyses were per-
formed with a 70 eV ionization potential. Electrospray-ionization
Figure 1. Monomeric units of eumelanin: 5,6-dihydroxyindole
DHI, 1) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA, 2).
Monomer of pheomelanin: cysteinyl-DOPA (3).
(
(
ESI) mass spectral data was performed in positive ESI mode. Melting
points are uncorrected.
,4-Dihydroxyphenylalanine benzyl ester hydrochloride (5a). 3,4-
Dihydroxyphenylalanine (4) (2.0 g, 10 mmol) was stirred with toluene-
-sulfonic acid monohydrate (1.9 g, 10 mmol) in 20 mL benzyl alcohol
3
isolation and handling difficulties, the structure of the pigment
remains poorly understood.
4
at room temperature. Toluene-4-sulfonyl chloride (2.3 g, 12 mmol)
was added and the solution was heated at 80ꢁC for 2 h. The solution
was cooled to room temperature, diluted with 200 mL CHCl and
3
Early model studies and theoretical considerations into the
oligomerization of these monomeric units suggested that the
preferred cross-linking positions for DHI (1) were the 3- and 7-
positions (17,18). More recently, dimers and trimers linked
through the 2-, 4- and 7-positions have been isolated (16,19–
washed with saturated sodium bicarbonate solution (3 · 100 mL). The
organic layer was dried (Na SO ) and concentrated to ca 100 mL. A
stream of hydrogen chloride was bubbled through the solution and the
2
4
resulting white precipitate was collected to yield 5a (1.15 g, 34%), m.p.
1
2
1). Panzella et al. have characterized DHI tetramers that they
178–181ꢁC (lit. [34] m.p. 191–193ꢁC). H NMR (500 MHz, CD
3
OD): d
7
.36–7.3 (5H, m, Bn), 6.70 (1H, d, J = 8.0 Hz, Ar-H5), 6.67 (1H, d,
J = 2.1 Hz, Ar-H2), 6.48 (1H, dd, J = 8.0, 2.1 Hz, Ar-H6), 5.22 (2H,
m, CO CH ), 4.23 (1H, t, J = 7.0 Hz, Ha), 3.09–3.01 (2H, m, Hb,b).
C NMR (125 MHz, CD OD): d 170.1 (C=O), 146.9, 146.3, 136.2,
129.76, 129.72, 129.66, 126.2, 121.8, 117.4, 116.8, 69.1 (CO CH ), 55.4
obtained by peroxidase ⁄ H O -induced oxidative coupling of
2
2
2
,4¢-linked DHI dimers (22). These researchers have also
investigated the pulse radiolytic oxidation of 2,2¢, 2,4¢ and
,7¢-bis indole dimers derived from DHI (23). On the other
2
2
13
3
2
2
+
2
hand, DHICA (2) is believed to undergo polymerization
through primarily the 4- and 7-positions (24–26). Both ionic
and radical mechanisms for the oligomerization of melanins
have been suggested, and two hypotheses have been proposed
for the structure of the eumelanin pigment: the first of which
suggests that eumelanin is a long chain linear co-polymer of
DHI (1) and DHICA (2) units (25–27), while the second
hypothesis proposed for the structure of eumelanin is that the
polymer consists of a compilation of discrete, highly conju-
gated, planar oligomeric units (i.e. dimer, trimer, tetramer,
etc.) (4,28,29). Kaxiras et al. have extended this hypothesis,
suggesting a structural model of melanin in which four DHI
units, linked at positions 2 and 7, form a cyclic tetramer with
an inner porphyrin ring (30).
Recently, eumelanin-like materials soluble in DMF and
DMSO have been synthesized by oxidative polymerization of
DOPA (4) using benzoyl peroxide (31–33). Taking a different
approach, we chose to investigate the solubility of synthetic
melanin containing lipophilic ester groups derived from
polymerization of the DHICA benzyl (6a) and octyl ester
4
(Ca), 36.9 (Cb). HRMS calcd for C16H18NO [M+H] 288.1233,
found 288.1235.
,6-Dihydroxyindole-2-carboxylic acid benzyl ester (6a). 3,4-Di-
hydroxyphenylalanine benzyl ester hydrochloride (5a) (162 mg,
0.5 mmol) was dissolved in 130 mL of pH 6 acetate buffer (0.2 M)
under a nitrogen atmosphere. Ceric ammonium nitrate (1.096 g,
5
2
1
mmol) dissolved in 20 mL of the same buffer was added. After
5 min sodium dithionite (0.68 g, 4 mmol) was added to the red–
brown mixture and the solution was stirred for a further 5 min. The
reaction mixture was extracted into ethyl acetate (3 · 100 mL). The
combined organic layers were dried (Na SO ), filtered and concen-
2 4
trated to 5 mL. The crude residue was adsorbed onto silica gel (2 g),
loaded onto a pad of silica gel (10 g) and eluted with EtOAc ⁄ hexane
(
dihydroxyindole-2-carboxylic acid benzyl ester (6a) (48.2 mg, 34%),
3:2). The yellow band was collected and evaporated to yield 3,4-
1
m.p. 168–173ꢁC (lit. [34] m.p. 179–180ꢁC). H NMR (500 MHz,
3 2
(CD ) SO): d 11.27 (1H, br d, J = 1.4 Hz, NH), 9.16 (1H, br s, OH),
8
.63 (1H, br s, OH), 7.46–7.33 (5H, m, Bn), 6.94 (1H, dd, J = 1.4,
.8 Hz, H3), 6.87 (1H, s, H4), 6.79 (1H, s, H7), 5.31 (2H, s, CO CH ).
SO): d 161.0 (C=O), 146.6, 142.3, 136.5,
33.1, 128.5, 127.9, 127.8, 124.4, 119.9, 107.9, 104.9, 96.9, 65.2. HRMS
0
2
2
13
3 2
C NMR (125 MHz, (CD )
1
+
calcd for C16
log e = 4.25) (lit. [34] 315 nm [log e = 4.27]).
,4-Dihydroxyphenylalanine octyl ester hydrochloride (5b). 3,4-Di-
4
H13NO [M] 283.0844, found 283.0847. kmax 330 nm
(
3
hydroxyphenylalanine (4) (1.0 g, 5.0 mmol) was stirred with toluene-4-
sulfonic acid (1.7 g, 10 mmol) and toluene-4-sulfonyl chloride (2.3 g,
12 mmol) in 1-octanol (20 mL) at 80ꢁC for 4 h. The solution was
cooled to room temperature, diluted with 100 mL Et O and washed
2
with saturated sodium bicarbonate solution (3 · 100 mL). Hydrogen
chloride gas was bubbled through the solution and the white
(
6b). It was expected that the pigment resulting from the
oxidative polymerization of such monomers would show
increased solubility in a range of common organic solvents,
allowing them to be characterized by conventional spectro-
scopic techniques, thus allowing us to probe the structure of
the final melanin pigment, and to investigate the early
polymerization products formed during this process. The
results reported here show that we can indeed prepare such
organic-soluble melanins, capable of being cast into thin films.
We have determined reaction rate constants for the oligomer-
ization and we have been able to identify the positions on the
indole nuclei at which cross-linking occur.
precipitate collected to yield 5b (1.5 g, 85%), m.p. 170–173ꢁC (lit.
1
[
35] m.p. 180ꢁC). H NMR (500 MHz, CD
3
OD): d 6.74 (1H, d,
J = 8.0 Hz, H5), 6.67 (1H, d, J = 2.1 Hz, H2), 6.56 (1H, dd,
J = 8.0, 2.1 Hz, H6), 4.2–4.16 (3H, m, Ca, CO CH ), 3.09–3.01
2H, m, Hb,b), 1.62–1.66 (2H, m, CO CH CH ), 1.30 (10H, br s,
2
2
(
2
2
2
13
5
CD
· CH
2
), 0.89 (3H, t, J = 6.8 Hz, CH
OD): d 170.2 (C=O), 146.8, 146.2, 126.2, 121.7, 117.3, 116.7, 67.5,
3
). C NMR (125 MHz,
3
55.4, 37.0, 32.9, 30.27, 30.26, 29.5, 26.9, 23.7, 14.4. HRMS calcd for
+
17 4
C H28NO [M+H] 310.2018, found 310.2023.