Short-lived quinonoid species from 5,6-dihydroxyindole dimers en route to eumelanin polymers: Integrated chemical, pulse radiolytic, and quantum mechanical investigation

Article abstract of DOI:10.1021/ja0650246

The transient species formed by oxidation of three dimers of 5,6-dihydroxyindole (1), a major building block of the natural biopolymer eumelanin, have been investigated. Pulse radiolytic oxidation of 5,5′,6,6′-tetrahydroxy-2,4′-biindolyl (3) and 5,5′,6,6′-tetrahydroxy-2,7′-biindolyl (4) led to semiquinones absorbing around 450 nm, which decayed with second-order kinetics (2k = 2.8 × 10⁹ and 1.4 × 10⁹ M^-1 s ^-1, respectively) to give the corresponding quinones (500-550 nm). 5,5′,6, 6′-Tetrahydroxy-2,2′-biindolyl (2), on the other hand, furnished a semiquinone (λ_max = 480 nm) which disproportionated at a comparable rate (2k = 3 × 10⁹ M ^-1 s^-1) to give a relatively stable quinone (λ_max = 570 nm). A quantum mechanical investigation of o-quinone, quinonimine, and quinone methide structures of 2-4 suggested that oxidized 2-4 exist mainly as 2-substituted extended quinone methide tautomers. Finally, an oxidation product of 3 was isolated for the first time and was formulated as the hydroxylated derivative 5 arising conceivably by the addition of water to the quinone methide intermediate predicted by theoretical analysis. Overall, these results suggest that the oxidation chemistry of biindolyls 2-4 differs significantly from that of the parent 1, whereby caution must be exercised before concepts that apply strictly to the mode of coupling of 1 are extended to higher oligomers.

Full text of DOI:10.1021/ja0650246

Published on Web 11/11/2006
Short-Lived Quinonoid Species from 5,6-Dihydroxyindole
Dimers en Route to Eumelanin Polymers: Integrated
Chemical, Pulse Radiolytic, and Quantum Mechanical
Investigation
†
†
‡
†
Alessandro Pezzella, Lucia Panzella, Orlando Crescenzi, Alessandra Napolitano,
§,|
§,⊥
⊥
,‡
Suppiah Navaratman, Ruth Edge, Edward J. Land, Vincenzo Barone,* and
Marco d’Ischia*,†
Contribution from the Departments of Organic Chemistry and Biochemistry and of Chemistry
and INSTM, UniVersity of Naples Federico II, Via Cintia, I-80126 Naples, Italy, and
Lennard-Jones Laboratories, School of Physical & Geographical Sciences, Keele UniVersity,
Staffordshire ST5 5BG, Free Radical Research Facility, Daresbury Laboratory, Warrington,
Cheshire WA4 4AD, and BioSciences Research Institute, UniVersity of Salford, Salford M5 4WT,
United Kingdom
Received July 24, 2006; E-mail: baronev@unina.it; dischia@unina.it
Abstract: The transient species formed by oxidation of three dimers of 5,6-dihydroxyindole (1), a major
building block of the natural biopolymer eumelanin, have been investigated. Pulse radiolytic oxidation of
5
,5′,6,6′-tetrahydroxy-2,4′-biindolyl (3) and 5,5′,6,6′-tetrahydroxy-2,7′-biindolyl (4) led to semiquinones
9
9
-1
absorbing around 450 nm, which decayed with second-order kinetics (2k ) 2.8 × 10 and 1.4 × 10 M
-
1
s
, respectively) to give the corresponding quinones (500-550 nm). 5,5′,6, 6′-Tetrahydroxy-2,2′-biindolyl
(
2), on the other hand, furnished a semiquinone (λmax ) 480 nm) which disproportionated at a comparable
9
-1
s
-1) to give a relatively stable quinone (λmax ) 570 nm). A quantum mechanical
rate (2k ) 3 × 10 M
investigation of o-quinone, quinonimine, and quinone methide structures of 2-4 suggested that oxidized
-4 exist mainly as 2-substituted extended quinone methide tautomers. Finally, an oxidation product of 3
2
was isolated for the first time and was formulated as the hydroxylated derivative 5 arising conceivably by
the addition of water to the quinone methide intermediate predicted by theoretical analysis. Overall, these
results suggest that the oxidation chemistry of biindolyls 2-4 differs significantly from that of the parent 1,
whereby caution must be exercised before concepts that apply strictly to the mode of coupling of 1 are
extended to higher oligomers.
drug-binding properties,4 susceptibility to redox changes,7
-6
Introduction
The oxidative polymerization of 5,6-dihydroxyindole (1) and
8
strong excited-state-phonon coupling, and amorphous electri-
cal switch behavior. The socioeconomic and biomedical
importance of eumelanins stems from their relevance to racial
pigmentation, skin photoprotection, sun tanning, and pigmentary
disorders, such as albinism, vitiligo, and melanoma. Despite
intense research efforts spanning over nearly a century, the
eumelanin structure remains poorly understood, because of the
chemical heterogeneity and the lack of well-defined physico-
9
related tyrosine-derived metabolites is a central, most elusive
process in the biosynthesis of eumelanins, the characteristic
pigments responsible for the dark colorations of human skin,
10
1
hair, and eyes. Eumelanins exhibit marked insolubility in all
11
solvents and are characterized by a unique set of physicochem-
ical properties, including broad-band monotonic absorption in
2
3
the UV-vis range, persistent free radical centers, metal- and
(
(
(
(
4) Bridelli, M. G.; Ciati, A.; Crippa, P. R. Biophys. Chem. 2006, 119, 137-
†
Department of Organic Chemistry and Biochemistry, University of
145.
5) Hong, L.; Liu, Y.; Simon, J. D. Photochem. Photobiol. 2004, 80, 477-
Naples Federico II.
‡
481.
Department of Chemistry and INSTM, University of Naples Federico
6) Samokhvalov, A.; Liu, Y.; Simon, J. D. Photochem. Photobiol. 2004, 80,
II. §
84-88.
Daresbury Laboratory.
University of Salford.
Keele University.
7) Ye, T., Hong, L., Garguilo, J., Pawlak, A., Edwards, G. S.; Nemanich, R.
|
J.; Sarna, T.; Simon, J. D. Photochem. Photobiol. 2006, 82, 733-777.
⊥
(8) Meredith, P; Riesz, J. Photochem. Photobiol. 2004, 79, 211-216.
(9) McGinness, J.; Corry, P.; Proctor, P. Science 1974, 183, 853-855.
(10) Prota, G.; d’Ischia, M.; Napolitano, A. In The Pigmentary System: Its
Physiology and Pathophysiology; Nordlund, J. J., Boissy, R. E., Hearing,
V. J., King, R. A., Ortonne, J. P., Eds.; Oxford University Press: New
York, 1998; Chapter 24, pp 307-332.
(
(
(
1) Prota, G. Melanins and Melanogenesis; Academic Press: San Diego, CA,
1
992.
2) Nighswander-Rempel, S. P.; Riesz, J.; Gilmore, J.; Bothma, J. P.; Meredith,
P. J. Phys. Chem. B 2005, 109, 20629-20635.
3) Seagle, B. L.; Rezai, K. A.; Gasyna, E. M.; Kobori, Y.; Rezaei, K. A.;
Norris, J. R., Jr. J. Am. Chem. Soc. 2005, 127,11220-11221.
(11) Prota, G. Fortschr. Chem. Org. Naturst. 1995, 64, 93-148.
15490
9
J. AM. CHEM. SOC. 2006, 128, 15490-15498
10.1021/ja0650246 CCC: $33.50 © 2006 American Chemical Society

Quinonoid Species from 5,6-Hydroxyindole Dimers
A R T I C L E S
indole quinone in aqueous solution to exist almost exclusively
as a single o-quinone tautomer with no significant absorption
in the range 400-600 nm, suggesting that second-order decay
of semiquinone may involve other pathways, e.g., dimerization,
in addition to disproportionation. Separate calculations have also
Figure 1. Tautomeric equilibria of the quinone of 1.
21
been performed on the semiquinone and the quinone of 1, as
chemical properties, preventing successful application of modern
spectroscopic techniques.
The role of the 5,6-dihydroxyindoles as eumelanin building
blocks has been known since 192712 and is illustrated by their
rapid conversion to black insoluble pigments following exposure
to oxidizing enzymes, UV radiation, and chemical oxidants or
even on standing at neutral physiologic pH.12 Interest in 5,6-
dihydroxyindole oxidation is also spurred by the prospects of
preparing polymeric materials with controlled structural proper-
ties and functionalities for high-technology applications such
as biosensors and photovoltaics.13
Chemical efforts to elucidate the early species formed in the
oxidation of 1 led to the isolation of a collection of dimers and
trimers which revealed a prevalent mode of coupling of the
indole monomer through the 2-, 4-, and 7-positions, as shown
by the structures of the dimers 2-4.14
2
2
23
well as on dimers and hypothetical oligomer structures.
2
3b
Recently, it has been suggested that dihydroxyindoles could
also form “square” dimers (with two parallel or antiparallel
indole units) and that such dimers, featuring two intermonomer
bonds, are stable with respect to dimers with a single bond.
However, experimental evidence supporting their actual forma-
tion is so far lacking. The calculated oligomeric spectra
suggested a eumelanin structure consisting of oligomeric indole
quinone substructures arranged as stacked graphitic-like sheets.2
Virtually nothing is known on the oxidation chemistry of
oligomers of 1: all attempts to investigate the species formed
beyond the dimer level were unsuccessful due to the complex
mixtures of products formed and their instability and insolubility.
A significant goal in this area would therefore be to identify
the first species generated from oxidation of the oligomer
intermediates, to determine their spectroscopic properties and
kinetics of decay, and to map their patterns of reactivity in
comparison with the parent heterocycle 1, in order to delineate
main reaction channels and to predict their relative importance.
As an initial step toward this goal, we have improved previous
procedures for the preparation of the three major dimers of 1,
viz., 2-4, and have investigated for the first time the absorption
properties of the transient semiquinones and quinones generated
by pulse radiolytic oxidation of 2-4.
4,25
Computational analysis and simulation of electronic absorp-
tion spectra of an array of candidate products allowed structural
assignment to the transient quinonoid species from oxidation
of these dimers.
The mechanism of indole dimerization is uncertain, but a
catechol-quinone interaction seems the most likely route on
the basis of product structures and quinone trapping experiments
Results and Discussion
15,16
with sulfur nucleophiles.
Valuable insights have also derived from the application of
pulsed radiation techniques. One-electron oxidation of 1 in
aqueous solution at pH 7.4, using the azide radical as an oxidant,
leads to an initial transient spectrum with peaks at 330 and 490
nm and a shoulder at 360 nm,17 ascribed to an oxygen-centered
radical with significant electron delocalization over the nitrogen
ring. This semiquinone may decay via disproportionation to
give a mixture of o-quinone, quinone methide, and quinonimine
species (Figure 1). It was argued on the basis of a peak in the
Preparation of Dimers 2-4. Although dimers 2-4 were
first described two decades ago by biomimetic oxidation of
1
4,26
1,
their preparation even on a milligram scale remains a
most difficult task. No concise organic synthesis for the
construction of the tetrahydroxybiindolyl systems has been
described so far, and oxidative coupling of 1 is to date the only
viable option. For the purposes of the present study, optimized
procedures for preparing small amounts of dimers 2-4 in pure
form by oxidation of 1 were set up, involving use of horseradish
peroxidase (HRP)/H2O2 in phosphate buffer at pH 7. Under these
conditions dimers 3 and 4 were obtained at a molar ratio of 1.6
and could be isolated as acetyl derivatives in better yields
1
8
4
30 nm region that the initial product observed after 5,6-
dihydroxyindole semiquinone disproportionation is the quinone
methide.19
However, quantum mechanical computations20 by methods
rooted in the density functional theory (DFT) predicted 5,6-
compared to those of previous procedures.
14
In the course of those studies, we found that addition of
organic solvents such as methanol, ethanol, THF, or acetone
(
12) Raper, H. S. Biochem. J. 1927, 21, 89-96.
(
13) de Albuquerque, J. E.; Giacomantonio, C.; White, A. G.; Meredith, P. Eur.
Biophys. J. 2006, 35, 190-195.
(21) Powell, B. J.; Baruah, T.; Bernstein, N.; Brake, K.; McKenzie, R. H.;
Meredith, P.; Pederson, M. R. J. Chem. Phys. 2004, 120, 8608-8615.
(22) Stark, K. B.; Gallas, J. M.; Zajac, G. W.; Eisner, M.; Golab, J. T. J. Phys.
Chem. 2003, 107, 3061-3067.
(23) (a) Stark, K. B.; Gallas, J. M.; Zajac G. W.; Eisner, M.; Golab, J. T. J.
Phys. Chem. 2003, 107, 11558-11562. (b) Tran, M. L.; Powell, B. J.;
Meredith, P. Biophys. J. 2006, 90, 743.
(24) Cheng, J.; Moss, S. C.; Eisner, M. Pigm. Cell Res. 1994, 7, 263-273.
(25) Zajac, G. W.; Gallas, J. M.; Alvarada-Swaisgood, A. E. J. Vac. Sci. Technol.,
B 1994, 12, 1512.
(26) Napolitano, A.; Corradini, M. G.; Prota, G. Tetrahedron Lett. 1985, 26,
2805-2808.
(
14) d’Ischia, M.; Napolitano, A.; Tsiakas, K. Prota, G. Tetrahedron 1990, 46,
5
789-5796.
(
15) d’Ischia, M.; Napolitano, A.; Prota, G. Tetrahedron 1987, 43, 5351-5356.
16) d’Ischia, M.; Napolitano, A.; Pezzella, A.; Land, E. J.; Ramsden,C. A. Riley,
P. A. AdV. Heterocycl. Chem. 2005, 89, 1-63.
(
(
17) Lambert, C.; Chacon, J. N.; Chedekel, M. R.; Land, E. J.; Riley, P. A.;
Thompson, A.; Truscott, T. G. Biochim. Biophys. Acta 1989, 993, 12-20.
18) Felix, C. C.; Sealy, R. C. Photochem. Photobiol. 1981, 34, 423-429.
19) Al-Kazwini, A.T.; O’Neill, P.; Adams, G. E.; Cundall, R.B.; Lang, G.;
Junino, B. J. Chem. Soc., Perkin Trans. 2 1991, 1941-1945.
(
(
(
20) Il’ichev, Y. V.; Simon, J. D. J. Phys. Chem. 2003, 107, 7162-7171.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006 15491

A R T I C L E S
Pezzella et al.
subverted the 3:4 product ratio in favor of the latter dimer: for
example, oxidation of 1 in acetone/0.1 M phosphate buffer, pH
7
(3:1, v/v), resulted in a 3 times higher formation yield of 4
(12% molar yield) with respect to 3.
Formation of 2 is known to depend on transition-metal
26
catalysis. Accordingly, an optimized procedure for the prepa-
ration of 2 was set up which involved HRP/H2O2 oxidation of
1
in â-hydroxy-4-(2-hydroxyethyl)-1-piperazinepropanesulfonic
acid (HEPPSO) buffer at pH 7.0 in the presence of 2.0 equiv
2+
of Ni , indicating that metal ions may exert a directional effect
by chelate formation with the o-diphenolic moiety of 1. Under
these conditions, dimers 3 and 4 were not formed in appreciable
amounts. Interestingly, acetone or ethanol virtually suppressed
the metal-induced formation of 2, suggesting that the chelate is
disrupted in the presence of organic solvents. It is very likely
that metal binding affects the equilibrium between the tautomeric
forms of the constituent monomers, but how this happens could
not be assessed at the present level of investigation and goes
beyond the scope of this study.
Figure 2. Changes in absorption at various times after pulse radiolysis of
-
4
a N2O-saturated aqueous solution of dimer 2 (1.5 × 10 M) in 0.5 M
-2
KBr/7.0 × 10 M phosphate buffer, pH 7.0: (O) 130 µs; (9) 480 µs; (2)
1.38 ms; ([) 4.4 ms.
Pulse Radiolysis. Pulse radiolysis was carried out as de-
scribed previously.2 Generation of the oxidizing species Br2
7,28
•-
was achieved by irradiating N2O-saturated solutions of 0.5 M
KBr. Under such conditions, Br2•- radicals are formed within
0
.1 µs after the radiation pulse according to the following
equations:
H O Df e- + OH
•
2
aq
e- + N O + H O f OH + N + OH
•
-
aq
2
2
2
Figure 3. Changes in absorption at various times after pulse radiolysis of
-
4
a N2O-saturated aqueous solution of dimer 3 (1.5 × 10 M) in 0.5 M
•
-
-
•
KBr/7.0 × 10-2 M phosphate buffer, pH 7.0: (O) 14 µs; (9) 102 µs; (2)
OH + Br f OH + Br
402 µs; ([) 866 µs.
•
-
•-
2
Br + Br f Br
Because dimers 2-4 were obtained and stored as the
tetraacetyl derivatives, an efficient deacetylation procedure
compatible with the experimental conditions for pulse radiolysis
was required. This procedure was set up using 5,6-diacetoxy-
indole as a reference compound and involved treatment of the
acetylated indole under a N2O atmosphere in 0.025 M trisodium
phosphate containing 0.5 M KBr, followed by addition of
sodium dihydrogen phosphate up to pH 7.0. To test the
efficiency of the procedure and to ascertain whether the
experimental conditions used in the present study allowed
comparison of data with previous results,17 5,6-diacetoxyindole
(
0.15 mM) was deacetylated by the above procedure and
Figure 4. Changes in absorption at various times after pulse radiolysis of
-
4
a N2O-saturated aqueous solution of dimer 4 (1.5 × 10 M) in 0.5 M
subjected to pulse radiolysis. The results were compared with
those obtained in another series of experiments using the parent
1
azide radical as the oxidizing agent. In both cases, a similar
semiquinone chromophore was obtained displaying a distinct
maximum at 480 nm, as reported,17 which changed into a species
with a less defined chromophore (460 nm with accompanying
broad shoulders in the 410-490 nm range) decreasing mono-
tonically with increasing wavelength.
-
2
KBr/7.0 × 10 M phosphate buffer, pH 7.0: (O) 18 µs; (9) 100 µs; (2)
02 µs; ([) 866 µs.
4
at the same concentration and in previous studies with the
irradiation of a N2O-saturated 0.15 mM solution of the substrate
that had been deacetylated under N2O just prior to irradiation.
The series of successive transient absorption spectra thus
obtained from 2-4 are shown in Figures 2-4, respectively. In
each case, the first transient spectrum was recorded at such a
time that the buildup of initial radical absorption was complete
and that subsequent decay had not proceeded to any significant
extent.
Figure 2 shows that the initial product from 2 completely
formed 130 µs after the pulse was the semiquinone, with a sharp
maximum around 480 nm and a broad, less intense band
centered at 650 nm. The rate constant for the reaction of 2 with
17
On the basis of these preliminary experiments, pulse radiolytic
one-electron oxidation of dimers 2-4 was carried out by
(
27) Napolitano, A.; Di, Donato, P.; Prota, G.; Land, E. J. Free Radical Biol.
Med. 1999, 27, 521-528.
(
28) Land, E. J.; Ramsden, C. A.; Riley, P. A. J. Photochem. Photobiol., B
2
001, 64, 123-135.
15492 J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006

Quinonoid Species from 5,6-Hydroxyindole Dimers
A R T I C L E S
Br2•- was estimated to be k ) 9 × 10 M s-1. The extinction
7
-1
(TDDFT)31 approach both in vacuo and in aqueous solution,
22
coefficient of the semiquinone at 480 nm was calculated to be
using the large 6-311++G(2d,2p) basis set. Previously, a DFT
analysis of five oxidized 5,6-dihydroxyindole dimers was
reported, using both local density approximation (LDA) and
generalized gradient approximation (GGA) functionals. In that
study, however, only doubly quinonoid structures, viz., at an
oxidation state two electrons higher than those described in the
present study, were considered. In particular two conformers
of a 3,7′-biindolyl system as yet devoid of experimental support
were investigated.
4
-1
-1
4
.0 × 10 M cm .
The transient species decayed by second-order kinetics (2k
9
-1 -1
)
3 × 10 M s ) to a species with a broad band centered at
4 -1 -1
5
70 nm (ꢀ ) 9.0 × 10 M cm ) and a shoulder at 520 nm.
The isosbestic point at 500 nm denoted direct conversion of
the first species to the bathochromically shifted chromophore.
It may be noted that the spectrum of the relatively stable species
formed from the decay of the semiquinone of 2 is closely similar
to that of melanochrome observed in the metal-catalyzed
oxidation of 2.26 Indeed, the species may well be identical.
Figures 3 and 4 indicate that 3 and 4 behaved quite differently
from 2. Dimer 3 gave a semiquinone (λmax ) 460 nm, ꢀ ) 11700
For the purposes of the present study, we chose to use the
“hybrid” PBE0 functional, which has been shown to provide
quite satisfactory energies and geometries for a wide range of
3
2
organic and biological systems, as well as a rather accurate
-
1
-1
9
-1
33
M
cm ) with a formation rate constant k ) 1.8 × 10 M
description of low-lying excited states. To assess the perfor-
-1
s . The semiquinone(s) apparently decayed with second-order
mance of the selected DFT technique in predicting electronic
absorption spectra of dimeric quinones, test calculations were
carried out on simple benzoquinones/benzoquinone imines, and
on indigo, a prototypical conjugated indole dimer. Overall, the
results (see the Supporting Information) confirmed that the
method chosen is capable of reproducing the UV-vis spectra
of such chromophores with reasonable accuracy. Thus, for
example, if one focuses on the most intense transitions, the
predicted wavelength for p-benzoquinone in vacuo is 247 nm,
to compare with an experimental value of 240 nm. For
p-benzoquinonimine (in water) the computed transition is at 266
nm, while the experimental spectrum shows a split band with
maxima at 260 and 254 nm. The related N-phenyl-p-benzo-
quinonimine in methanol has strong transitions at 490, 309, and
267 nm, in fair agreement with the experimental values of 448,
285, and 263 nm. A comparable error is obtained in the case of
indigo (computed 570 nm, measured 600 nm).
9
-1 -1
kinetics (2k ) 2.8 × 10 M s ), resulting in the generation
of species with broad bands at 490 nm (ꢀ ) 17700 M cm )
and at 530 nm (ꢀ ) 15900 M cm ). Dimer 4 gave a
-
1
-1
-
1
-1
9
-1 -1
semiquinone (formation rate constant k ) 2.2 × 10 M s )
with a broader, seemingly split band (λmax ) 430 nm, ꢀ ) 8.6
3
-1
-1
3
-1
-1
×
10 M cm , and 480 nm, ꢀ ) 8.1 × 10 M cm ). The
semiquinone(s) decayed with second-order rate constants 2k )
9
-1 -1
9
1
M
.4 × 10 M
s
measured at 480 nm and 2k ) 2.1 × 10
-
1
-1
s
determined at 430 nm. Concomitant to semiquinone
decay, a chromophore developed with a broad maximum
centered around 530 nm. If it is assumed that a single
semiquinone species is responsible for the bands at 420 and
4
80 nm, then the extinction coefficient of the quinone can be
-1
-1
calculated to be 11100 M cm at 530 nm taking as reference
the semiquinone band at 480 nm. If there is more than one
quinone, such a calculation is invalid. Overall, these pulse
radiolysis studies suggested that oxidation of 2-4 leads to the
generation of semiquinone intermediates which decay by a
bimolecular process. This may consist of either disproportion-
ation, to give two-electron oxidation products generically
referred to as “quinones”, or dimerization, to afford tetraindolyl
species. To address this issue, a theoretical investigation of the
main tautomeric forms of quinones from dimers 2-4 was
undertaken.
Theoretical Studies and Simulated Electronic Absorption
Spectra of Biindolyl Quinone Tautomers. Pairs of syn-
periplanar (sp) and anti-periplanar (ap) rotamers can be envis-
aged for the o-quinone, quinone methide, and quinonimine
tautomers of dimers 2-4, depending on the orientation of the
indole rings around the single bond, whereas two geometrical
isomers (E and Z) are possible for the extended forms. To
determine which of these structures may be a plausible candidate
for the observed two-electron oxidation products of the dimers,
The molecular structures of the main quinone tautomers for
dimer 2 along with their relative energies are shown in Figure
5; a complete listing of the structures examined is presented in
the Supporting Information.
DFT results suggest that the extended forms 2a and 2b are
by far the most stable tautomers in the series. The structures
that come next, namely, methides 2c and 2d in vacuo, and
quinone 2g in aqueous solution, are separated by several
kilocalories per mole. Interestingly, in vacuo the E isomer 2b
is planar and slightly more stable than the Z isomer 2a, in which
the indole rings are twisted by 3.4°. However, in aqueous
solution both isomers are planar and the E isomer is destabilized
-
1
by ca. 1.1 kcal mol . With respect to all other tautomers, the
o-quinone tautomers feature one less intramolecular OH-O
hydrogen bond. Thus, their relative stability increases signifi-
(
31) (a) Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. J. Chem. Phys. 1998,
1
09, 8218-8224. (b) Bauernschmitt, R.; Ahlrichs, R. Chem. Phys. Lett.
all tautomeric quinones of dimers 2-4 were geometry-optimized
1996, 256, 454-464. (c) Casida, M. E.; Jamorski, C.; Casida, K. C.;
Salahub, D. R. J. Chem. Phys. 1998, 108, 4439-4449. (d) Adamo, C.;
Scuseria, G. E.; Barone, V. J. Chem. Phys. 1999, 111, 2889-2899.
in vacuo at the PBE0/6-31+G(d,p) level of theory.29 For each
-
1
dimer, all structures that were within ca. 15 kcal mol from
the minimum-energy structure were also optimized using the
polarizable continuum model (PCM)30 to simulate the aqueous
environment; the absorption spectra of all the energetically
relevant species were computed using the time-dependent DFT
(32) (a) Improta, R.; Barone, V. J. Am. Chem. Soc. 2004, 126, 14320-14321.
(
b) Benzi, C.; Improta, R.; Scalmani, G.; Barone, V. J. Comput. Chem.
2
002, 23, 341-350. (c) Langella, E.; Rega, N.; Improta, R.; Crescenzi,
O.; Barone, V. J. Comput. Chem. 2002, 23, 650-661. (d) Improta, R.;
Mele, F., Crescenzi, O.; Benzi, C.; Barone, V. J. Am. Chem. Soc. 2002,
1
24, 7857-7865.
(33) (a) Jacquemin, D.; Wathelet, V.; Perpete, E. A. J. Phys. Chem. A 2006,
10, 9145-9152. (b) Preat, J.; Jacquemin, D.; Wathelet, V.; Andre, J.-M.;
1
Perpete, E. A. J. Phys. Chem. A 2006, 110, 8144-8150. (c) Jacquemin,
D.; Preat, J.; Wathelet, V.; Fontaine, M.; Perpete, E. A. J. Am. Chem. Soc.
2006, 128, 2072-2083. (d) Crescenzi, O.; Pavone, M.; De Angelis, F.;
Barone, V. J. Phys. Chem. B 2005, 109, 445-453. (e) Aquilante, F.; Cossi,
M.; Crescenzi, O.; Scalmani, G.; Barone, V. Mol. Phys. 2003, 101, 1945-
1953. (f) Adamo, C.; Barone, V. Chem. Phys. Lett. 2000, 330, 152-160.
(
29) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158-6170.
(
30) (a) Miertus, S.; Scrocco, E.; Tomasi, J. Chem. Phys. 1981, 55, 117-129.
(
b) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002,
1
17, 43-54. (c) Scalmani, G.; Barone, V.; Kudin, K. N.; Pomelli, C. S.;
Scuseria, G. E.; Frisch, M. J. Theor. Chem. Acc. 2004, 111, 90-100.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006 15493

A R T I C L E S
Pezzella et al.
unaltered (see the Supporting Information), the energy gaps to
other tautomers tend to become uniformly smaller; in particular,
-
1
the contributions of 2c (at 0.8 kcal mol ) and 2i (at 1.1 kcal
-1
mol ) would become significant. Computed absorption spectra
of these latter isomers, along with those of 2a and 2b, are given
in Table 1. For a comparison of all computed absorption spectra
with experimental data see the Supporting Information.
The computed absorption maxima of the main components
2
a and 2b are quite close: around 500 nm in a vacuum and
bathochromically shifted to ca. 550 nm in aqueous solution.
These results are in qualitative agreement with the experimen-
tally measured absorption maximum (570 nm) of the species
formed by second-order decay of the transient semiquinone from
2.
Figure 5. Structures and relative energies (kcal mol-1) of several tautomers/
conformers of 2, computed at the PBE0/6-31+G(d,p) level in vacuo or in
aqueous solution (PCM). In parentheses the inter-ring N-C-C-N dihedral
angle (deg) and/or the symmetry group of the molecule is reported.
As to the nature of the bands in 2a and 2b, the DFT results
(in vacuo) show that the 500 nm band corresponds to an allowed
transition (501 nm, f ) 1.625, and 512 nm, f ) 1.672,
respectively) with a strong HOMO f LUMO character. Longer
wavelength transitions are also predicted, albeit with much lower
oscillator strengths, corresponding essentially to HOMO - 1
f LUMO (575 nm, f ) 0.0005, for 2a, and 609 nm, f ) 0.014,
for 2b) and HOMO - 2 f LUMO (549 nm, f ) 0.056, for 2a,
and 571 nm, f ) 0.0000, for 2b) excitations. Except for the
wavelength shifts hinted above, these results are essentially
unaltered in aqueous solution.
cantly in an aqueous environment: however, this effect is not
sufficient to bring them close enough to the extended tautomers.
In this dimer series, alternative conformations differing only in
the orientation of the OH groups of a dihydroxyindole ring (e.g.,
2c and 2d) were separately optimized. Within each pair, the
conformer that orients the OH groups opposite to the nitrogen
is invariably more stable; therefore, in the following only this
conformation was considered, unless a preliminary inspection
suggested that a different hydrogen bond pattern could arise in
the other orientation. The above calculations therefore identified
The extended quinone methide structure 3a (Figure 6),
featuring a 2-substituted quinone methide moiety, was predicted
to be the most stable of the tautomer structures under consid-
eration for the “quinone” of dimer 3 (see the Supporting
Information). In particular, the Z isomer was stabilized relative
2
a as the primary determinant of the measured absorption
spectrum of 2 quinone, with only a minor contribution from
the E isomer 2b (ca. 15%, based on energy differences).
Inclusion of harmonic vibrational contributions (see the Sup-
porting Information) has a marginal impact on the overall
picture. As a further check, single-point energies in aqueous
solution were also computed at the MP2/6-31+G(d,p)/PCM
level. While the separation between 2a and 2b is basically
-1
to the E isomer both in vacuo (by 5.1 kcal mol ) and in aqueous
-1
solution (by 3.1 kcal mol ), due to a hydrogen bond that forms
between the indole NH group and the adjacent carbonyl oxygen
atom. The next most stable tautomer was the sp 2-substituted
Table 1. Transition Wavelengths (nm) and Oscillator Strengths Computed at the PBE0/6-311+G(2d,2p)//PBE0/6-31+G(d,p) Level for the
Most Stable Tautomers of 2-4 in Vacuo and/or in Aqueous Solution (PCM)a
In Vacuo
2a
549 (0.056), 501 (1.625), 301 (0.015), 286 (0.032)
2b
609 (0.014), 512 (1.672)
2
c
744 (0.014), 505 (0.146), 486 (0.702), 326 (0.104), 305 (0.029), 297 (0.464)
884 (0.037), 492 (0.621), 343 (0.197), 332 (0.180), 284 (0.025)
815 (0.062), 604 (0.013), 470 (0.679), 330 (0.093), 328 (0.208)
756 (0.016), 511 (0.428), 338 (0.266), 305 (0.134), 294 (0.156), 289 (0.054)
884 (0.073), 529 (0.483), 361 (0.299), 334 (0.165), 284 (0.011)
819 (0.100), 584 (0.030), 504 (0.499), 352 (0.263), 331 (0.180)
748 (0.013), 551 (0.332), 357 (0.357), 352 (0.035), 308 (0.071), 293 (0.104), 282 (0.054)
3a
3e
3
i
4a
4e
4
j
In Aqueous Solution
2a
589 (0.160), 551 (1.742), 358 (0.018), 287 (0.070)
2b
659 (0.140), 558 (1.758)
2
c
761 (0.051), 577 (0.021), 527 (1.123), 335 (0.149), 310 (0.032), 300 (0.394)
828 (0.188), 547 (0.016), 522 (0.960), 332 (0.330), 319 (0.052), 292 (0.143)
792 (0.158), 553 (0.115), 529 (0.871), 328 (0.323), 315 (0.127), 294 (0.093), 282 (0.047)
956 (0.047), 655 (0.014), 534 (0.722), 357 (0.271), 340 (0.192), 291 (0.018), 282 (0.021)
929 (0.071), 524 (0.781), 349 (0.214), 339 (0.185), 313 (0.017)
2
3
g
2
a
i
3e
3
i
817 (0.029), 763 (0.013), 552 (0.516), 353 (0.367), 314 (0.189), 301 (0.100), 294 (0.035)
826 (0.081), 652 (0.049), 534 (0.377), 338 (0.256), 320 (0.086), 302 (0.107), 294 (0.169), 286 (0.077)
897 (0.124), 557 (0.573), 395 (0.011), 373 (0.323), 338 (0.191), 295 (0.019)
871 (0.153), 550 (0.594), 369 (0.322), 337 (0.209)
3p
4a
4e
4
j
818 (0.022), 766 (0.016), 577 (0.424), 366 (0.458), 311 (0.086), 297 (0.136), 283 (0.046)
804 (0.077), 663 (0.019), 554 (0.309), 348 (0.314), 310 (0.111), 300 (0.114), 292 (0.196), 283 (0.020)
779 (0.111), 670 (0.017), 557 (0.264), 347 (0.319), 310 (0.116), 298 (0.137), 293 (0.170)
4o
4q
a
Only transitions with a wavelength above 280 nm and an oscillator strength above 0.010 are reported.
1
5494 J. AM. CHEM. SOC. VOL. 128, NO. 48, 2006
9

Quinonoid Species from 5,6-Hydroxyindole Dimers
A R T I C L E S
Figure 6. Structures and relative energies (kcal mol-1) of several tautomers/
conformers of 3, computed at the PBE0/6-31+G(d,p) level in vacuo or in
aqueous solution (PCM). In parentheses the inter-ring N-C-C-C(O)
dihedral angle (deg) is reported.
quinone methide 3i, in which a hydrogen bond can form between
the nitrogen of the quinone methide ring and an OH group of
the 4-substituted ring. This structure, however, was less favor-
Figure 7. Structures and relative energies (kcal mol-1) of several tautomers/
conformers of 4, computed at the PBE0/6-31+G(d,p) level in vacuo or in
aqueous solution (PCM). In parentheses the inter-ring N-C-C-C(O)
dihedral angle (deg) is reported.
-
1
-1
able by 6.2 kcal mol in a vacuum and by 7.4 kcal mol in
aqueous solution compared to 3a, suggesting that it should not
be present to an appreciable extent. The aqueous environment
preferentially stabilizes the o-quinones (e.g., 3p), but even so
none of them reach a stability comparable to that of the extended
tautomers. Again, single-point computations at the MP2/PCM
level (see the Supporting information) showed a general
flattening of the potential energy surface, but in this case no
conformer comes close enough to the absolute minimum to
become significantly populated.
The computed absorption spectra of quinones from 3 are
given in Table 1. Calculations predicted for the most stable
tautomer 3a and for the E isomer 3e in aqueous solution
absorption maxima around 530 and 520 nm, respectively. Thus,
both stability and excitation energy data suggest that tautomers
predominantly localized on the 2-substituted ring. The computed
intensity of longer wavelength bands is at least a factor of 10
smaller: the HOMO f LUMO transition is predicted at 884
nm in 3a (815 nm for 3e), while the HOMO - 2 f LUMO
transition would be at 634 nm (respectively 604 nm). The nature
and ordering of these long-wavelength transitions are unchanged
in aqueous solution, apart from wavelength shifts.
Figure 7 presents the results of DFT computations for
tautomers of 4. Again, the extended quinone methide 4a,
featuring a 2-substituted quinone methide moiety, was the most
stable tautomer, whereas the E isomer 4e was disfavored by
-1
-1
5
.6 kcal mol in vacuo and 2.8 kcal mol in aqueous solution.
The next most stable tautomers, e.g., the 2-substituted quinone
methide 4j and the 2-substituted o-quinones 4q and 4p, were
less favorable by several kilocalories per mole in aqueous
solution in comparison to 4a and are not expected to contribute
to the spectrum. The picture is unaltered at the MP2/PCM level
3
a and 3e are responsible for the 530 nm band observed in the
radiolysis experiments (Figure 3).
Since it is known that the PCM model can show limitations
in the accurate description of highly specific solute-solvent
interactions, the computed UV-vis spectrum of 3a was used
as a test case to evaluate the results of a cluster/PCM approach,
(see the Supporting Information).
The most intense bands in the electronic absorption spectra
which has proved very versatile in reproducing static and
of 4a and 4e in aqueous solution were predicted to be around
50 nm (Table 1) and were in satisfactory agreement with the
absorption maxima measured in the radiolysis experiments
dynamic solvation effects.3
3d,34
Thus, TDDFT/PCM computa-
5
tions were performed on some water adducts of 3a, in which
an explicit water molecule was hydrogen-bonded either to a
carbonyl or to a hydroxyl group. However, in the wavelength
region of interest the transitions turned out to be almost
unchanged with respect to those computed with PCM alone (see
the Supporting Information). Such insensitivity to the exact
details of the hydrogen-bonding geometry is probably related
to the nature of the transitions, which involve mainly strongly
delocalized π orbitals.
(Figure 4).
The nature of the electronic transitions underlying the UV-
vis spectrum is similar to that for the 2,4-dimer: the main band
529 nm, f ) 0.483, for 4a in vacuo; 504 nm, f ) 0.499, for
e) is mostly HOMO - 1 f LUMO in character, while the
HOMO f LUMO (4a, 884 nm, f ) 0.073; 4e, 819 nm, f )
(
4
0
5
.100) and HOMO - 2 f LUMO (4a, 634 nm, f ) 0.007; 4e,
84 nm, f ) 0.030) transitions are much less intense. Also the
From examination of the TDDFT results (in vacuo), the
principal bands of 3a and 3e correspond to HOMO - 1 f
LUMO excitations (3a, 492 nm, f ) 0.621; 3e, 470 nm, f )
basic features of the molecular orbitals are similar to those in
the other asymmetric dimer, with the HOMO and to a lesser
extent the HOMO - 1 mostly localized on the 7-substituted
ring, while the HOMO - 2 and the LUMO are predominantly
on the 2-substituted ring.
0.679). In this case, the HOMO - 1 molecular orbital is mostly
localized on the 4-substituted indole ring, while the LUMO is
It is noteworthy that both 4a and 4e show a bathochromic
shift of about 30 nm in their main band, with respect to the
analogous tautomers 3a and 3e; shifts are much less prominent
for the other low-energy transitions. As hinted above, the shifted
(
34) (a) Barone, V.; Crescenzi, O.; Improta, R. Quant. Struct.- Act. Relat. 2002,
2
1, 105-118. (b) Cossi, M.; Crescenzi, O. J. Chem. Phys. 2003, 118, 8863-
8
872. (c) Pavone, M.; Benzi, C.; De, Angelis, F.; Barone, V. Chem. Phys.
Lett. 2004, 395, 120-126. (d) Ciofini, I.; Adamo, C.; Barone, V. J. Chem.
Phys. 2004, 121, 6710-6718.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006 15495

A R T I C L E S
Pezzella et al.
Figure 8. Isodensity surfaces (0.04 au) of selected molecular orbitals of
Figure 9. Proposed mechanism of formation of 5 by oxidation of dimer 3.
3
a and 4a, computed at the PBE0/6-311++G(2d,2p)//PBE0/6-31+G(d,p)
level in vacuo.
in a separate set of experiments the oxidation of 3 by the HRP/
H2O2 system in 0.05 M phosphate buffer, pH 7, was investi-
gated. Dimer 3 was selected because of its prevalent formation
in the oxidation of 1 under biomimetic conditions, which makes
it a most plausible intermediate in the eumelanin pathway in
vitro and in vivo. Moreover, its asymmetric structure was
expected to represent a more useful probe to investigate dimer
quinone reactivity and to obtain proof of the putative extended
quinone methide intermediate.
transition has a dominant HOMO - 1 f LUMO character.
Figure 8 shows a comparison of the involved orbitals in 3a and
4
a: while the LUMOs are quite similar, the HOMO - 1
molecular orbitals extend across the two rings, with significant
coefficients on the nitrogen atoms of the 4- and, respectively,
7
-substituted indole moieties. Therefore, it is reasonable to
expect that a change in the position of the linkage may
preferentially affect the energy of this orbital. The situation is
perfectly parallel for 3e vs 4e.
TLC and HPLC analysis after acetylation and the usual
workup indicated a very complex mixture of species difficult
to separate. One of the products eluted under a band at Rf )
From the above calculations, it is concluded that extended
quinone methides are by far the most stable among biindolyl
quinone tautomers. The satisfactory agreement between experi-
mental and simulated spectra allows moreover the conclusion
that the products arising from second-order decay of semiquino-
nes are the quinones rather than tetrameric species. In the case
of asymmetric dimers 3 and 4, the most favorable among minor
tautomers, in which conjugation does not extend beyond a single
ring, are usually those in which the oxidized moiety is the
0
.36 (eluant CHCl3-MeOH, 98:2) proved more amenable to
chromatographic isolation and was eventually obtained in minute
+
amounts (less than 3%) for spectral characterization. The ESI -
MS spectrum gave a pseudo molecular ion peak at m/z 545 [M
+
+
Na] , suggesting a pentaacetoxybiindolyl. Extensive 2D NMR
analysis consistently indicated five acetyl groups and the lack
of the H-3 proton in the 2-substituted ring. A noticeable feature
of the proton NMR spectrum was an unusually downfield singlet
at δ 8.20 ascribed to the H-4 proton in the 2-substituted unit
experiencing the deshielding effect of the adjacent acetyl group
2
-substituted indole ring.
As an additional remark, it has been noted that continuum
models may fail to properly account for highly specific solute-
solvent interactions (e.g., H-bonds). These interactions are
apparently not crucial for the UV-vis spectra in the present
case, as shown by the cluster calculations reported above.
However, they could still play a relevant role in the determi-
nation of the relative stabilities of the different isomers. A
systematic exploration of all water-dimeric quinone clusters
would clearly be extremely expensive: therefore, we resorted
to performing test cluster-PCM calculations on the parent
indole quinone tautomers, whereby one water molecule from
the first solvation shell is explicitly included in several possible
optimized positions. The results (see the Supporting Information)
indicate that the relative stability of a specific cluster is affected
by the exact positioning of the explicit water molecule, but that
overall the energetic trend of the different quinone tautomers
is well reproduced by simple PCM computations (i.e., without
explicit waters), thus corroborating the use of the continuum
model to assess relative stabilities.
(
see Table 1S in the Supporting Information). On this basis,
the product was formulated as the pentaacetyl derivative of the
novel 3,5,5′,6,6′-pentahydroxy-2,4′-biindolyl (5).
The remainder of the reaction mixture consisted apparently
of tetramers of 1 and higher oligomers (LC-MS evidence),
which will form the subject of a separate study.
Although product 5 is clearly not fully representative of the
complex reactivity pattern of 3 quinone, its isolation is nonethe-
less of interest because it reveals hitherto overlooked aspects
of 5,6-dihydroxyindole oxidation chemistry remarkably pre-
dicted by theoretical calculations. Available evidence indicates
that indole 1 does not tend to react through the 3-position, the
only relevant precedent being the isolation of an adduct at C-3
between 1 and the antithyroid drug thiouracil. However,
introduction of an additional OH group in the case of 5 reflects
an unexpected electrophilic reactivity of 3 quinone at the
hindered 3-position of the 2-substituted indole moiety. This is
possible only if that position is rendered electrophilic by a double
3
5
Isolation and Characterization of an Oxidation Product
from Dimer 3. To gain experimental evidence for the involve-
ment of extended quinone methide tautomers in dimer oxidation,
(35) Napolitano, A.; Palumbo, A.; d’Ischia, M.; Prota, G. J. Med. Chem. 1996,
39, 5192-5201.
15496 J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006

Quinonoid Species from 5,6-Hydroxyindole Dimers
A R T I C L E S
bond efficiently conjugated with a carbonyl function within a
planar structural moiety. Though a number of structures can be
envisaged, it is noteworthy that the extended quinone methide
intermediates 3a and 3e fulfill at best this requirement. A
plausible mechanism accounting for formation of 5 is depicted
in Figure 9.
Experimental Section
,6-Dihydroxyindole was synthesized as previously reported.38 For
general experimental methods and other materials see the Supporting
Information.
Preparation of Dimers 2-4. A solution of 1 (300 mg, 2.0 mmol)
in 0.05 M phosphate buffer (120 mL) was treated with HRP (36 U/mL)
5
2 2
and H O (266 µL of a 30% solution, 2.3 mmol). After 25 s of reaction
time, the oxidation mixture was halted by the addition of a solution of
Isolation of product 5 moreover corroborates the previous
hypothesis17,36 that quinones decay via reaction of the methide
with water, generating hydroxylated species. This mechanism
may also offer a likely explanation to the long-standing issue
sodium dithionite in water up to a 50 mM final concentration and
14
worked up as reported. After acetylation with acetic anhydride-
pyridine overnight at room temperature the mixture was fractionated
1
,37
of oxygen incorporation during eumelanin buildup.
by column chromatography (gradient elution, CHCl -AcOEt from 10%
3
to 40%) to afford 3 (70 mg, 15% yield, >90% pure) and 4 (45 mg,
1
out in acetone/0.1 M phosphate buffer, pH 7.0, 1:1, v/v, using HRP
Conclusions. We have provided herein the first insight into
the transient intermediates generated by oxidation of 5,6-
dihydroxyindole dimers and have underscored the methodologi-
cal potential of chemical, pulse radiolytic, and quantum
mechanical approaches when used in an integrated fashion.
Spectrophotometric characterization of the species formed from
pulse radiolytic oxidation and structural assignments to relevant
chromophores have been carried out with the aid of a systematic
computational analysis of all tautomers compatible with two-
electron oxidation structures and simulation of their electronic
absorption spectra. These efforts resulted in a consistent picture
of tautomerization equilibria for the quinones of dimers 2-4
that highlighted the extended quinone methides as the most
stable tautomers for all biindolyl quinones investigated. Upon
oxidation, the nonplanar geometries of 2-4 are converted to
extended quinone methide structures in which the double bond
constrains the biindolyl system to an approximately planar
geometry, possibly more amenable to be assembled into a
stacked layer polymeric architecture. In this frame, it would
also be of interest to extend calculations to the “square” dimers
hypothesized by previous authors,2 to verify their relevance
with the already considered systems, and this will be addressed
in a separate study.
0% yield, >95% pure).14 In other experiments the reaction was carried
(
72 U/mL) and H (266 µL of a 30% solution, 2.3 mmol). After 80
2 2
O
s of workup and fractionation of the oxidation mixture as above afforded
the acetyl derivatives of dimers 3 (18 mg, 4% yield) and 4 (58 mg,
13% yield), respectively. Dimer 2 (as the O-acetyl derivative, 30 mg,
12% yield)26 was obtained by oxidation of 1 (150 mg, 1.0 mmol) in
0
2
.05 M HEPPSO buffer, pH 7.5, containing NiSO
.0 mmol).
4
2
‚7H O (560 mg,
Note: Formation yields of dimers 2-4 are very sensitive to the
reaction conditions, especially the time of oxidation of 1 and mode of
addition of reagents. Isolated yields reported above are averages of at
least three separate optimized preparations, with deviations not exceed-
ing (25%. Failure to adhere to the above protocol may result in
significant variations in dimer formation yields.
Pulse Radiolysis. The pulse radiolysis experiments were performed
with the 12 MeV linear accelerator at the Daresbury Laboratory, using
the Free Radical Research Facility.39 This accelerator provides pulse
lengths of between 0.2 and 2 µs with doses up to 30 Gy using quartz
capillary cells of optical path length 2.5 cm. Absorbed doses were
•
-
determined from the transient (CNS)
2
formation in air-saturated
potassium thiocyanate solutions (10 mM) using a G of 0.30 µM/Gy
3b
-1
-1 40
and ꢀ (500 nm) ) 7100 M cm
.
The estimates of the molar
absorption coefficients and rate constants are considered to be correct
to (15%. In a typical experiment, a solution of the appropriate indole
as an acetyl derivative in methanol (15 mM) was treated under a N
atmosphere with a solution of 0.025 M trisodium phosphate containing
.5 M KBr up to a 0.15 mM concentration, and after 1 min the pH of
the solution was taken to 7.0 by addition of NaH PO (0.07 M final
2
O
The isolation of pentahydroxybiindolyl 5, an unprecedented
hydroxylated indole derivative by oxidation of dimer 3, is
another important outcome of the present study. It not only
provides convincing experimental evidence for the proposed
generation of the extended quinone methide intermediates, but
also reveals a significant difference between the oxidation
chemistry of 1 and its dimers.
0
2
4
concentration of phosphate buffer). The resulting mixture was subjected
to pulse radiolysis.
Isolation of Dimer 5 as a Pentaacetyl Derivative. A solution of 3
(93 mg, 0.2 mmol) in methanol (5.0 mL) was taken under a flux of
argon and treated with 0.1 M phosphate buffer, pH 12 (24.0 mL), that
As far as the impact of this study on the process of eumelanin
buildup in vivo is concerned, it is clear that the strictly anaerobic
pulse radiolysis conditions, favoring the transient formation of
high semiquinone concentrations, differ from those expected
during natural melanogenesis. However, such conditions do
allow sufficient amounts, not otherwise attainable, of the
quinonoid products of radical disproportionation to permit their
direct detection spectroscopically. It cannot be ruled out that
during eumelanin synthesis in the biological environment of
melanocytes other reactions may become important, such as,
for example, the interaction of molecular oxygen with transient
free radicals, which should be the subject of future further
studies.
2 4
had prior been purged with argon. After 1 min solid KH PO was added
to pH 7.4, and peroxidase (1800 pyrogallol units) was added followed
by 30% hydrogen peroxide (80 µL, 4 molar equivalents). After about
20 s the reaction was stopped by addition of a 0.2 M solution of sodium
dithionite in water (4 mL) and acidified to pH 3 by addition of 2 M
HCl. The mixture was extracted with ethyl acetate (3 × 30 mL), and
the combined organic layers were dried over sodium sulfate and taken
to dryness. The residue was acetylated with acetic anhydride-pyridine,
9
3
5:5, v/v, and fractionated by preparative TLC (CHCl /MeOH, 98:2)
to give 5 as a pentaacetyl derivative (3 mg, 3% yield, R
f
) 0.36): HR
(
39) (a) Butler, J.; Hodgson, B. W.; Hoey, B. M.; Land, E. J.; Lea, J. S.; Lindley,
E. J.; Rushton, F. A. P.; Swallow, A. J. Radiat. Phys. Chem. 1989, 34,
6
33-646. (b) Holder, D. J.; Allan, D.; Land, E. J.; Navaratnam, S. In
Proceedings of the 8th European Particle Accelerator Conference; Garvey,
T., Le Duff, J., Le Roux, P., Petit-Jean-Genaz, C., Poole, J., Rivki, L.,
Eds.; European Physical Society: Paris, 2002; pp 2804-2806.
(
36) Lambert, C.; Land, E. J.; Riley, P. A.; Truscott, T. G. Biochim. Biophys.
(40) (a) Buxton, G. V.; Stuart, C. R. J. Chem. Soc., Faraday Trans. 1995, 91,
279-281. (b) Adams, G. E.; Boag, J. W.; Currant, J.; Michael, B. D. In
Pulse Radiolysis; Ebert, M., Keene, J. P., Swallow, A. J., Baxendale, J.
H., Eds.; Academic Press: London, 1965; pp 117-129.
Acta 1990, 1035, 319-324.
(
37) Nicolaus, R. A. In Melanins; Lederer, E., Ed.; Hermann: Paris, 1968.
38) Benigni, J. D.; Minnis, R. L. J. Heterocycl. Chem. 1965, 2, 387-392.
(
J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006 15497

A R T I C L E S
Pezzella et al.
+
+
ESI -MS m/z found 545.1156 [M + Na] , m/z calcd for C26
H N
22 2
O
10
-
pulse radiolysis experiments were carried out at the Free Radical
Research Facility (Station 0.1) in the Synchrotron Radiation
Department of the CCLRC Daresbury Laboratory, Warrington,
U.K., and were financially supported by EC Grant No. 43332.
We thank the Centro Interdipartimentale di Metodologie Chimico-
Fisiche (CIMCF; University of Naples Federico II) for use of
its NMR and mass spectrometry facilities. The technical
assistance of Mrs. Silvana Corsani is gratefully acknowledged.
1
13
Na 545.1172. H and C NMR data are reported in Table 1S.
Computational Methods. All calculations were performed with the
Gaussian 03 package of programs. Geometries were optimized at the
DFT level of theory using the PBE0 functional with the 6-31+G(d,p)
basis set. PBE0 (also referred to as PBE1PBE) is a hybrid functional
obtained by combining a predetermined amount of exact exchange with
the Perdew-Burke-Ernzerhof exchange and correlation functionals.
4
1
4
2
29
To simulate the aqueous environment, the PCM30 was used, in
combination with the UAHF parametrization for atomic radii.43 All
minima were checked by computing the harmonic vibrational frequen-
cies. MP2/PCM single-point energies were computed with the same
basis set for comparison purposes. Electronic absorption spectra of
significant tautomers were estimated on the basis of excitation energy
calculations using the TDDFT approach. The PBE0 functional with
the large 6-311++G(2d,2p) basis set was used for TDDFT computa-
tions, either in the gas phase or in solution with the PCM method. In
the latter case, the linear response nonequilibrium model was used.44
To produce graphs of computed UV-vis spectra, transitions above 190
nm were selected, and an arbitrary Gaussian line width of 50 nm was
imposed.
Supporting Information Available: General methods, NMR
spectral data for dimer 5, complete ref 41, computed UV-vis
spectra of model quinone compounds, structures, relative
energies, and UV-vis spectra of tautomers/conformers of 2-4
computed at the PBE0/6-31+G(d,p) level in vacuo or in aqueous
solution, relative energies of selected tautomers/conformers of
2-4, computed at the MP2/6-31+G(d,p) and PBE0/6-31+G-
3
1
(d,p) levels in aqueous solution (PCM), relative free energies
of selected tautomers/conformers of 2-4, computed at the
PBE0/6-31+G(d,p) level in vacuo and in aqueous solution
(PCM), transition wavelengths and oscillator strengths computed
at the PBE0/6-311+G(2d,2p)/PCM//PBE0/6-31+G(d,p)/PCM
level for 3a with and without the inclusion of one explicit
hydrogen-bonded water molecule at a given position, and
kinetics of pulse radiolytic oxidation of compounds 2-4 at
different wavelengths. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. We thank the CampusGrid of the Uni-
versity Federico II of Naples for computational resources. The
(
41) Frisch, M. J.; et al. Gaussian 03, Revision C.02; Gaussian, Inc.: Walling-
ford, CT, 2004.
42) Francl, M. M.; Petro, W. J.; Hehre, W. J. S.; Binkley, J.; Gordon, M. S.;
DeFrees, D. J.; Pople, J. A. J. Chem. Phys. 1982, 77, 3654-3665. For a
general introduction to basis sets see: Foresman, J. B.; Frisch, A. Exploring
Chemistry with Electronic Structure Methods, 2nd ed.; Gaussian, Inc.:
Pittsburg, PA, 1996.
(
(
(
43) Barone, V.; Cossi, M.; Tomasi, J. J. Chem. Phys. 1997, 107, 3210-3221.
44) Cossi, M.; Barone, V. J. Phys. Chem. A 2000, 104, 10614-10622.
JA0650246
15498 J. AM. CHEM. SOC.
9
VOL. 128, NO. 48, 2006