Versatile Effects of Aurone Structure on Mushroom Tyrosinase Activity

Article abstract of DOI:10.1002/cbic.201100716

Elucidation of the binding modes of Ty inhibitors is an important step for in-depth studies on how to regulate tyrosinase activity. In this paper we highlight the extraordinarily versatile effects of the aurone structure on mushroom Ty activity. Depending on the position of the OH group on the B-ring, aurones can behave either as substrates or as hyperbolic activators. The synthesis of a hybrid aurone through combination of an aurone moiety with HOPNO (2-hydroxypyridine N-oxide), a good metal chelate, led us to a new, efficient, mixed inhibitor for mushroom tyrosinase. Another important feature pointed out by our study is the presence of more than one site for aurone compounds on mushroom tyrosinase. Because study of the binding of the hybrid aurone was difficult to perform with the enzyme, we undertook binding studies with tyrosinase functional models in order to elucidate the binding mode (chelating vs. bridging) on a dicopper(II) center. Use of EPR combined with theoretical DFT calculations allowed us to propose a preferred chelating mode for the interaction of the hybrid aurone with a dicopper(II) center.

Full text of DOI:10.1002/cbic.201100716

DOI: 10.1002/cbic.201100716
Versatile Effects of Aurone Structure on Mushroom
Tyrosinase Activity
Carole Dubois,[a] Romain Haudecoeur,[b] Maylis Orio,[c] Catherine Belle,[c] Constance Bochot,[c]
Ahcꢀne Boumendjel,[b] Renaud Hardrꢁ,[a] Hꢁlꢀne Jamet,[c] and Marius Rꢁglier*[a]
Dedicated to Andreas Heumann on the occasion of his 70th birthday
Elucidation of the binding modes of Ty inhibitors is an impor-
tant step for in-depth studies on how to regulate tyrosinase
activity. In this paper we highlight the extraordinarily versatile
effects of the aurone structure on mushroom Ty activity. De-
pending on the position of the OH group on the B-ring, au-
rones can behave either as substrates or as hyperbolic activa-
tors. The synthesis of a hybrid aurone through combination of
an aurone moiety with HOPNO (2-hydroxypyridine N-oxide), a
good metal chelate, led us to a new, efficient, mixed inhibitor
for mushroom tyrosinase. Another important feature pointed
out by our study is the presence of more than one site for
aurone compounds on mushroom tyrosinase. Because study of
the binding of the hybrid aurone was difficult to perform with
the enzyme, we undertook binding studies with tyrosinase
functional models in order to elucidate the binding mode (che-
lating vs. bridging) on a dicopper(II) center. Use of EPR com-
bined with theoretical DFT calculations allowed us to propose
a preferred chelating mode for the interaction of the hybrid
aurone with a dicopper(II) center.
Introduction
Tyrosinases (Ty’s, EC 1.14.18.1) are ubiquitous copper-contain-
ing enzymes belonging to the type-3 or “coupled binuclear”
family.[1,2] Ty’s catalyze the two-step oxidation of phenolic com-
pounds into the corresponding catechols (monophenolase ac-
tivity) and ortho-quinones (catecholase activity). In mammals,
their biological function is to convert l-tyrosine into dopaqui-
none, which is the key product for melanin pigment biosyn-
HOPNO compound embedded in an aurone backbone. In this
paper we report on the evaluation of aurone derivatives 1a–e
on mushroom Ty activity and on the synthesis of the hybrid
aurone 2 and its evaluation as an inhibitor of mushroom Ty.
thesis. Melanin-related disorders are known to cause serious Results and Discussion
skin lesions,[3] Parkinson’s disease,[4] and melanoma.[5] In addi-
Versatile effects of aurones on mushroom Ty activity
tion, Ty’s are responsible for the browning of plant foods,
which creates an important economic problem in the field of
nutrition. Ty inhibition is a well-established approach for con-
trolling in vivo melanin production and food browning, so the
development of Ty inhibitors has a huge economical and in-
dustrial impact, as is illustrated by the increasing number of Ty
inhibitors described in the recent literature.[6] A large propor-
tion of this literature is devoted to naturally occurring com-
pounds. Among these, aurones are particularly attractive be-
cause they have been reported as inhibitors of melanin synthe-
sis in human melanocytes.[7] Some of these naturally occurring
Ty inhibitors target the Ty binuclear copper active site. These
inhibitors are named transition state (TS) analogues; they have
structural analogies to catechol but are not oxidizable. Thuja-
plicin,[8] kojic acid,[9] and l-mimosin[10] are good illustrations of
this strategy. Recently, we reported on 2-hydroxypyridine-N-
oxide (HOPNO) as a non-naturally occurring TS analogue Ty
inhibitor with a competitive inhibition constant of 1.8 mm.[11]
Because HOPNO is well known as good metal chelate,[12] we
might suspect some lack of specificity, which could limit its
use. In order to improve the specificities of HOPNO moieties
towards Ty, we investigated the synthesis and evaluation of a
In a previous study, the most potent aurones were found to be
those bearing a hydroxy group at the 4’-position of the B-
ring.[7] In the belief that aurones interact with the binuclear
copper active site through this ring, we made a study of au-
rones in which the position of the hydroxy group on the A-
[a] C. Dubois,+ Dr. R. Hardrꢀ, Dr. M. Rꢀglier
Institut des Sciences Molꢀculaires de Marseille, ꢁquipe BiosCiences
UMR-CNRS 7313, Aix–Marseille Universitꢀ
avenue Escadrille Normandie-Niemen
13397 Marseille Cedex 20 (France)
E-mail: marius.reglier@univ-amu.fr
[b] R. Haudecoeur,+ Prof. A. Boumendjel
Dꢀpartement de Pharmacochimie Molꢀculaire, Universitꢀ Joseph Fourier
UMR-CNRS 5063, ICMG FR-2607
B.P. 53, 38041 Grenoble Cedex 9 (France)
[c] Dr. M. Orio,+ Dr. C. Belle, Dr. C. Bochot, Dr. H. Jamet
Dꢀpartement de Chimie Molꢀculaire, ꢁquipes Chimie Thꢀorique et CIRE
Universitꢀ Joseph Fourier
UMR-CNRS 5250, ICMG FR-2607
B.P. 53, 38041, Grenoble Cedex 9 (France)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100716.
ChemBioChem 2012, 13, 559 – 565
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
559

M. Rꢀglier et al.
ring is fixed in the 6-position and the one on the B-ring varies
(compounds 1a–e). The effect of compounds 1a–e on Ty-cata-
lyzed l-DOPA oxidation revealed the versatile effects of the
aurone structure.
sity of the band at 400 nm and concomitant increase in the
one at 499 nm, together with the two isosbestic points at 348
and 446 nm, indicate a transformation into an unique com-
pound that should be a quinone resulting from oxidation of
phenol either on the A- or on the B-ring.
Under the same conditions, the catechol aurone 1e also
undergoes a transformation as revealed by a decrease in the
intensity of the band at 414 nm with an isosbestic point at
367 nm (Figure S2).
Because of their instability, all of our attempts to character-
ize products formed during the Ty-catalyzed oxidation of au-
rones 1b and 1e failed. We overcame this problem by addition
of NaBH3CN at the end of the reaction in order to reduce the
formed quinone into a more stable catechol (Scheme 1). HPLC
analysis (C18, 3% AcOH in water/acetonitrile) of the reduced
mixtures revealed the formation of aurone 1e (30%) during
Ty-catalyzed oxidation of 1b, as confirmed with an authentic
sample of 1e (Figure S3).
In this study, we used a commercially available mushroom
Ty that we purified on a Q-sepharose FF chromatographic
column by a known procedure.[13] For Ty-catalyzed l-DOPA oxi-
dation, we determined the Michaelis–Menten kinetic parame-
ters—Km =0.31ꢀ0.6 mm and V=0.87ꢀ0.06 mmsꢁ1—by meas-
uring the increase in absorbance at 475 nm, which corre-
sponds to the formation of the DOPAchrome (Figure S1 in the
Supporting Information).
Whereas aurone 1a exhibits no activity, the others behave
either as substrates (1b and 1e) or as hyperbolic activators (1c
and 1d), depending on the position(s) of the hydroxy group(s)
on the B-ring. The UV–visible spectra of aurones 1a–e were
checked in the presence of mushroom Ty. This experiment re-
vealed that the para-phenol 1b undergoes a transformation as
a result of Ty-catalyzed oxidation. In the UV–visible spectrum
(300–600 nm, Figure 1) of aurone 1b in phosphate buffer
(pH 7, 50 mm) containing Ty (3.3 mg), the decrease in the inten-
Scheme 1. Ty-catalyzed oxidations of the para-phenol 1b and the catechol
1e and quinone trapping by NaBH3CN.
For the two substrates 1b and 1e, we determined the kinet-
ic features of the Ty-catalyzed oxidation by measuring the de-
creases in the absorption bands at 400 nm. The kinetics of Ty-
catalyzed oxidation of aurones 1b and 1e exhibit non-Michae-
lis–Menten behavior with sigmoid shapes according to a coop-
erative mechanism (Figure 2). A classical treatment according
to the Hill equations[14] [Eqs. (1), (2), and (3)] leads to the
macroscopic kinetic features V, K0.5, and h (Hill coefficients) for
these two substrates [1b, V=(0.40ꢃ10ꢁ3 ꢀ0.02ꢃ10ꢁ3
)
DODsꢁ1, K0.5 =365ꢀ32 mm, and h=2.05ꢀ0.22; 1e, V=(2.21ꢃ
10ꢁ3 ꢀ0.05ꢃ10ꢁ3) DODsꢁ1 and K0.5 =28ꢀ2 mm and h=1.87ꢀ
0.17].
V ꢂ Sh
V ¼
ð1Þ
ð2Þ
Kh0:5 þ Sh
Sh
Figure 1. Absorption spectra of 1b (4.37 mm) in phosphate buffer (50 mm,
pH 7) in the presence of mushroom Ty (3.3 mg). Spectra were scanned every
minute until 5 min.
V
Vꢁv
¼
Kh0:5
560
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ChemBioChem 2012, 13, 559 – 565

Tyrosinase Effectors
Figure 3. Hyperbolic activation of mushroom Ty by aurones 1c and 1d. Con-
ditions: phosphate buffer (50 mm, pH 7), [l-DOPA]=2.8 mm, [Ty]=
6.4 mgmLꢁ1
.
ures of cooperativity in binding processes, indicate that the
binding of one ligand facilitates the binding of a subsequent
ligand at the catalytic site. This proposal is supported by the
Ty activation by aurones that are not substrates—the meta-
phenol 1c and ortho-phenol 1d. These molecules interact with
Ty at at least one site, which increases the Ty activity. Activa-
tion of latent Ty from plant and insect sources is well known,
but is rare for mushroom Ty.[6] Activation of mushroom Ty by
SDS,[15] benzyl alcohol,[16] 3-hydroxyanthranilic acid,[17] and 3-hy-
droxykynurenine[18] has been reported. This activation is often
attributed to conformational changes on Ty or to removal of
an inhibitor during the purification. The proposal that aurone
compounds could interact with mushroom Ty at at least two
sites is particularly important because the recently published
structure confirms that mushroom Ty exists as a H2L2 tetra-
mer.[19] However, we cannot definitively exclude the possibility
that these effects could be due to the presence of Ty isoforms
with different kinetic features.
Figure 2. Plots of initial rates of Ty-catalyzed oxidation of A) 1b, and B) 1e
vs. substrate concentrations (fitting according to the Hill equation, goodness
of fit R=0.99641 and 0.99711, respectively); Hill plots in insets. Conditions:
phosphate buffer (50 mm, pH 7) in the presence of mushroom Ty
(6.4 mgmLꢁ1).
ꢀ
ꢁ
V
Vꢁv
log
¼ h ðlog Sꢁlog K0:5
Þ
ð3Þ
Although the UV–visible spectra of meta-phenol 1c and ortho-
phenol 1d were not modified by Ty-catalyzed oxidation (Fig-
ure S4), they behave as activators in the presence of the sub-
strate l-DOPA. Indeed, they exhibit dose-dependent activation
in Ty-catalyzed oxidation of l-DOPA (1c, 214% and 1d, 118%
activation at 300 mm for [l-DOPA]=2.8 mm; Figure 3B). Classi-
cal treatment involving plotting of 1/Vapp against reciprocal
activator concentration showed that the aurones 1c and 1d
behave as hyperbolic activators (Figure 3A).[14]
The cooperativity in Ty-catalyzed oxidations of aurones 1b
and 1e suggests that they interact with mushroom Ty at at
least two different sites: a catalytic site and one allosteric site
that increases the enzyme activity. The Hill coefficients, meas-
ChemBioChem 2012, 13, 559 – 565
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561

M. Rꢀglier et al.
HOPNO embedded on aurone—synthesis and mushroom Ty
inhibition
With the goal of finding new Ty inhibitors, we combined
aurone recognition by mushroom Ty with the inhibition poten-
tial of HOPNO. Because we have shown that the aurones inter-
act with the dinuclear copper active site through their B-rings,
we prepared the new aurone 2 in which the B-ring is replaced
by a HOPNO moiety. The synthesis of aurone 2 (Scheme 2) was
Scheme 2. Synthesis of aurone 2: a) KOH, CH3OH, 658C, 2 h, 93%; b) Ac2O,
1408C, 5 h, 96%; c) H2O2/urea, TFAA, CH2Cl2, RT, 16 h, 63%; d) HCl, CH3OH,
1008C, 16 h, 83%.
performed by starting from 6-hydroxybenzofuran-3(2H)-one,
prepared by the known reported method.[20] It was condensed
with 6-methoxypyridine-3-carbaldehyde, followed by protec-
tion of the phenol groups. N-Oxidation with H2O2/urea adduct
and deprotection under acidic conditions afforded the aurone
2, which was obtained with a good overall yield and a high
analytical purity.
The effect of aurone 2 on the oxidation of l-DOPA by mush-
room Ty was studied. We first determined that the Ty inhibi-
tion was concentration-dependent, with an IC50 estimated as
1.5ꢀ0.2 mm (Figure S5). The kinetic behavior of mushroom Ty
during the oxidation of l-DOPA was then investigated. Aurone
2 exhibits mixed inhibition features, as revealed by a graphical
method consisting of plotting 1/v (Dixon plot) and [l-DOPA]/v
against the inhibitor concentration (Figure S6). The macroscop-
ic kinetic features for the competitive inhibition (Kic) and the
uncompetitive inhibition (Kiu) were determined by evaluation
of Vapp and Kampp from the Michaelis–Menten plot at different
concentrations of 2 (Figure 4a).[14] From Equation (4) and the
1/Vapp versus [2] plot, we extracted Kiu =1.62ꢀ0.28 mm, where-
as from Equation (5) and the Kampp/Vapp vs. [2] plot it was deter-
mined that Kic =1.27ꢀ0.16 mm.
Figure 4. Inhibition of mushroom Ty by aurone 2. Conditions: phosphate
buffer (50 mm, pH 7), [Ty]=6.4 mgmLꢁ1. Dependence: A) of the initial rate (vi)
~
!
&
*
^
on [l-DOPA] at various [2] ( =0, =0.2, =1, =2, =5, =20 mm),
B) of 1/Vapp on [2], and C) of Kmapp/Vapp on [2].
ꢂ
ꢃ
Kampp Km
I
¼
ꢂ
1 þ
ð5Þ
V app
V
Kic
Aurone 2 has an activity similar to that of HOPNO (Kic =
1.8 mm),[11] but with different features (competitive vs. mixed in-
hibition). However, with aurone 2 we can expect an increase in
the selectivity with respect to Ty. The mixed inhibition features
ꢂ
ꢃ
1
V app
1
V
I
¼
ꢂ
1 þ
ð4Þ
Kiu
562
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ChemBioChem 2012, 13, 559 – 565

Tyrosinase Effectors
of 2 again suggest a multiple binding site for this derivative.
The deciphering of its binding mode is an important step for
studies on how to regulate the Ty activity.
Binding studies
The evaluation of the inhibition properties of 2 gives evidence
that this compound interacts in part with the Cu ions in the
binuclear active site of the enzyme. We set out to study the
interaction of 2 with mushroom Ty by UV–visible spectroscopy.
Unfortunately, the addition of 2 to mushroom Ty produced no
observable change in the UV–visible spectrum, and so we ori-
ented our study on the interaction of 2 on Ty complex models.
A first approach to obtain more insight into its binding mode
at the Ty active site was to study inhibitor interactions with a
low-molecular-weight binuclear copper system mimicking the
dicopper(II) center of the enzyme, combining experimental
and theoretical investigations. In this way, through the use of
the binuclear copper(II) complex [Cu2(BPMP)(m-OH)](ClO4)2
[I·(ClO4)2], known as a functional model for the Ty catecholase
activity,[21] we have previously shown that deprotonated
HOPNO chelates only one Cu atom of the binuclear site in a
chelating mode (Scheme 3).[11]
Figure 5. Graphical representation of the binding modes (2-bg: bridging, 2-
ch: chelating) for adducts of complex I2+ with 2.
are available for I2+ +2, the DFT-predicted structures of the
two binding modes are compared exclusively on the basis of
the computational results. The chelating mode shows an elon-
gated Cu–Cu interatomic distance relative to the bridging
mode. Consequently, the exchange interaction between the
two copper centers becomes
less efficient and a weak ferro-
magnetic coupling (J=12 cmꢁ1)
is calculated for this mode. On
the other hand, the two Cu cen-
ters in 2-bg are antiferromagnet-
ically coupled (J=ꢁ255 cmꢁ1).
These data, in conjunction with
the EPR spectrum mentioned
before, suggest that 2 is linked
to I2+ in a chelating mode. To
confirm this result, we compared
Scheme 3. [Cu2(BPMP)(m-OH)](ClO4)2 as a functional model for Ty catecholase activity and possible binding modes
for 2 at the dicopper(II) center.
the relative stabilities of the two
modes with the aid of a compu-
tational approach described in
the Supporting Information. Re-
Unfortunately, attempts to isolated some adducts with 2 in
the solid state have so far been unsuccessful. Nevertheless, the
X-band EPR spectrum of [I·(ClO4)2] after the addition of 1 equiv
of 2 in frozen solution [HEPES (pH 7)/DMSO 1:1] suggests a
change in copper coordination geometry and supports the
binding of 2 at the dicopper center (Figure S7). The spectrum
reveals a weak DMsꢀ2 transition around 150 mT and a broad
DMsꢀ1 signal from 230 to 400 mT, which is characteristic of
slightly magnetically coupled copper(II) ions, whereas [I·(ClO4)2]
is EPR-silent, in accordance with the relatively strong antiferro-
magnetic coupling between the copper ions [J=(ꢁ112 ꢀ
sults of the energetic analysis are shown in Figure 5. Our calcu-
lations predict that 2-ch should be the more favored binding
mode, with an energetic stabilization of 16.0 kcalmolꢁ1. This is
the same binding mode that we found for HOPNO on I2+ [11]
.
Conclusions
Deciphering the binding modes of Ty inhibitors is an important
step for in-depth studies on how to regulate the tyrosinase ac-
tivity. Our results highlight the extraordinarily versatile effects
of the aurone structure on mushroom Ty activity. Depending
on the position of the hydroxy group on the B-ring, aurones
can behave as substrates (1b and 1e), as hyperbolic activators
(1c and 1d), or—in the particular case of the hybrid aurone
2—as a mixed inhibitor. This last compound has been identi-
fied as a new and efficient inhibitor for mushroom Ty. Another
important feature highlighted in our study is the presence of
multiple binding sites for aurone compounds on mushroom
2) cmꢁ1, with H=ꢁ2 JS1·S2; S1 =S2 =1= ).[21]
2
In order to get some information on the I2+ +2 binding
mode, theoretical calculations were undertaken. The two differ-
ent binding modes shown in Scheme 3 were considered and
possible I2+ +2 adducts formed are shown in Figure 5. They
are designated 2-bg and 2-ch, for the bridging and the chelat-
ing modes, respectively. Because no X-ray experimental data
ChemBioChem 2012, 13, 559 – 565
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563

M. Rꢀglier et al.
1H; 2’-H), 8.23 (dd, J1 =8.7 Hz, J2 =2.4 Hz, 1H; 6’-H), 7.82 (d, J=
8.3 Hz, 1H; 4-H), 7.14 (d, J=1.8 Hz, 1H; 7-H), 6.97 (dd, J1 =8.3 Hz,
J2 =1.8 Hz, 1H; 5-H), 6.85 (d, J=8.7 Hz, 1H; 5’-H), 6.85 (s, 1H;
ꢁCH=), 4.01 (s, 3H; OMe), 2.37 ppm (s, 3H; OAc); 13C NMR
(100 MHz, CDCl3): d=182.9 (C-3), 168.4 (C-6), 166.5 (C-8), 164.7
(OCOMe), 157.3 (C-4’), 150.9 (C-2’), 147.0 (C-2), 140.3 (C-6’), 125.6
(C-4), 121.9 (C-1’), 119.3 (C-9), 117.6 (C-5), 111.6 (ꢁCH=), 110.1 (C-5’),
106.7 (C-7), 53.8 (OMe), 21.2 ppm (OCOMe); MS (ESI): m/z: 312
[M+H]+, 334 [M+Na]+; elemental analysis calcd (%) for
Ty. In addition to the active site where substrates bind, at least
one other site could be responsible for activation due to modi-
fication of interactions between the H and L subunits of mush-
room Ty.
Because the study of the binding of 2 was difficult to carry
out on mushroom Ty, we undertook binding studies with Ty
functional models in order to elucidate the binding mode (che-
lating or bridging) on a dicopper(II) center. With the aid of a
combined EPR study with theoretical DFT calculations, we pro-
posed a preferred chelating mode (2-ch) for the interaction of
aurone 2 with a dicopper(II) center. These results are of pri-
mary importance because with this set of structural data (2-bg
vs. 2-ch) we can envisage an EXAFS/XANES study in order to
decipher the binding mode of aurones and then propose new
scaffolds that might better target the Ty metal active site for
more selective and potent inhibitor development.
C17H13NO5·1= H2O: C 64.66, H 4.28, N 4.44; found: C 64.18, H 4.02, N
4
4.49.
(Z)-6-Acetoxy-2-((6-methoxy-1-oxypyridin-3-yl)methylene)benzo-
furan-3(2H)-one (5): Urea hydrogen peroxide adduct (1.28 g,
12.88 mmol) was added at 08C to a solution of (Z)-6-acetoxy-2-((6-
methoxypyridin-3-yl)methylene)benzofuran-3(2H)-one
4
(1 g,
3.22 mmol) in dichloromethane (40 mL), followed by the addition
of trifluoroacetic anhydride (1.79 mL, 12.88 mmol). The mixture
was stirred at room temperature for 16 h and was then diluted
with dichloromethane, washed successively with sodium thiosul-
fate, water, and brine, dried over magnesium sulfate, and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (dichloromethane/methanol, 98:2
then 90:10 gradient elution) to afford the title compound (658 mg,
Experimental Section
Syntheses: 1H and 13C NMR spectra were recorded with a Brꢄker
1
Advance 400 instrument (400 MHz for H, 100 MHz for 13C). Chemi-
1
63%) as a yellow solid: m.p. 155–1578C; H NMR (400 MHz, CDCl3):
cal shifts are reported relative to Me4Si as internal standard. ESI
mass spectra and elemental analyses were performed at the analy-
sis facilities of the department of chemistry of the University of
Grenoble, France. Thin-layer chromatography (TLC) was carried out
with Merck silica gel F-254 plates (0.25 mm thick). Flash chroma-
tography was carried out with Merck silica gel 60, 200–400 mesh.
All solvents were distilled prior to use. Chemicals and reagents
were obtained either from Aldrich or Acros and were used as ob-
tained.
d=8.95 (d, J=2.0 Hz, 1H; 2’-H), 7.77 (d, J=8.3 Hz, 1H; 4-H), 7.65
(dd, J1 =8.7 Hz, J2 =2.0 Hz, 1H; 6’-H), 7.13 (d, J=1.8 Hz, 1H; 7-H),
6.97 (dd, J1 =8.3 Hz, J2 =1.8 Hz, 1H; 5-H), 6.96 (d, J=8.7 Hz, 1H; 5’-
H), 6.60 (s, 1H; ꢁCH=), 4.13 (s, 3H; OMe), 2.35 ppm (s, 3H; OAc);
13C NMR (100 MHz, CDCl3): d=182.8 (C-3), 168.4 (C-6), 166.8 (C-8),
158.9, 157.9 (C-4’, OCOMe), 148.0 (C-2), 141.5 (C-2’), 130.4 (C-6’),
125.9 (C-4), 123.5 (C-1’), 118.8 (C-9), 118.4 (C-5), 107.7 (C-5’), 107.0
(C-7), 106.6 (ꢁCH=), 57.7 (OMe), 21.4 ppm (OCOMe); MS (ESI): m/z:
328 [M+H]+, 350 [M+Na]+; elemental analysis calcd (%) for
C17H13NO6·1= H2O: C 60.71, H 4.16, N 4.16; found: C 60.74, H 4.08, N
(Z)-6-Hydroxy-2-((6-methoxypyridin-3-yl)methylene)benzofuran-
3(2H)-one (3): An aqueous solution of potassium hydroxide (50%,
7 mL) was added to a solution of 6-hydroxybenzofuran-3(2H)-one
(963 mg, 6.42 mmol) in methanol (100 mL), followed by the addi-
tion of 6-methoxypyridine-3-carbaldehyde (880 mg, 6.42 mmol).
The mixture was heated at reflux for 2 h, and the methanol was
then evaporated. The residue was diluted in water and a solution
of aqueous hydrochloric acid (10%) was added to adjust the pH to
2–3. The resulting precipitate was filtered and washed successively
with water, acetonitrile, and diethyl ether to afford hydrochloride
salt of the title compound (1.82 g, 93%) as a pale yellow solid:
m.p. 260–2628C (decomp.); 1H NMR (400 MHz, [D6]DMSO): d=
11.25 (s, 1H; OH), 8.66 (s, 1H; 2’-H), 8.28 (d, J=8.0 Hz, 1H; 6’-H),
7.60 (d, J=8.0 Hz, 1H; 4-H), 6.93 (d, J=8.0 Hz, 1H; 5’-H), 6.79 (s,
1H; ꢁCH=), 6.79 (s, 1H; 7-H), 6.71 (d, J=8.0 Hz, 1H; 5-H), 3.89 ppm
(s, 3H; OMe); 13C NMR (100 MHz, [D6]DMSO): d=181.1 (C-3), 167.8
(C-6), 166.6 (C-8), 163.8 (C-4’), 150.4 (C-2’), 147.2 (C-2), 140.4 (C-6’),
126.0 (C-4), 122.1 (C-1’), 113.1 (C-9), 112.9 (C-7), 111.2 (ꢁCH=), 107.4
(C-5’), 98.7 (C-5), 53.6 ppm (OMe); MS (ESI): m/z: 270 [M+H]+; ele-
2
4.41.
(Z)-6-Hydroxy-2-((6-hydroxy-1-oxypyridin-3-yl)methylene)benzo-
furan-3(2H)-one (2): An aqueous solution of hydrochloric acid
(10%, 4 mL) was added to a solution of (Z)-6-acetoxy-2-((6-meth-
oxy-1-oxypyridin-3-yl)methylene)benzofuran-3(2H)-one (5, 400 mg,
1.22 mmol) in methanol (6 mL) and the mixture was heated at
reflux for 16 h. After cooling, the precipitate was filtered and
washed successively with water, methanol, and diethyl ether to
afford the title compound (274 mg, 83%) as a white solid: m.p.
1
>2808C; H NMR (400 MHz, DMSO): d=12.19 (brs, 1H; OH), 11.17
(s, 1H; OH), 8.56 (s, 1H; 2’-H), 8.08 (d, J=9.2 Hz, 1H; 6’-H), 7.59 (d,
J=8.2 Hz, 1H; 4-H), 6.78 (s, 1H; 7-H), 6.72 (s, 1H; ꢁCH=), 6.65–
6.70 ppm (m, 2H; 5,5’-H); 13C NMR (100 MHz, DMSO): d=180.7 (C-
3), 167.3 (C-6), 166.3 (C-8), 157.0 (C-4’), 146.1 (C-2), 139.5 (C-2’),
138.6 (C-6’), 125.8 (C-4), 119.4 (C-1’), 113.0 (C-7,9), 110.6 (C-5’), 107.2
(ꢁCH=), 98.6 ppm (C-5); MS (ESI): m/z: 270 [MꢁH]ꢁ; elemental anal-
ysis calcd (%) for C14H9NO5·1= H2O: C 60.98, H 3.45, N 5.08; found:
4
mental analysis calcd (%) for C15H11NO4·3= HCl: C 60.73, H 3.96, N
C 60.73, H 3.19, N 5.22.
4
4.72; found: C 60.54, H 4.30, N 4.80.
Enzymatic assays: Mushroom tyrosinase (6.4 mg, purchased from
Sigma) was dissolved in phosphate buffer (pH 7.0, 50 mm, 5 mL)
and purified on Q-Sepharose FF with use of a gradient of NaCl
from 0 to 1.0m.[13] Purity of the tyrosinase was checked by SDS-
PAGE; the purified tyrosinase exhibits only two bands at MW
ꢃ14 kDa and 45 kDa. The tyrosinase activity was checked spectro-
scopically with use of l-DOPA as substrate (Figure S1).
(Z)-6-Acetoxy-2-((6-methoxypyridin-3-yl)methylene)benzofuran-
3(2H)-one (4): A solution of (Z)-6-hydroxy-2-((6-methoxypyridin-3-
yl)methylene)benzofuran-3(2H)-one (3, 378 mg, 1.41 mmol) in
acetic anhydride (8 mL) was heated at reflux for 5 h. After cooling,
the mixture was poured into water (60 mL) and was then extracted
with dichloromethane, washed with water and brine, dried over
magnesium sulfate, and concentrated under reduced pressure to
afford the title compound (419 mg, 96%) as a pale yellow solid:
m.p. 190–1928C; 1H NMR (400 MHz, CDCl3): d=8.60 (d, J=2.4 Hz,
All compounds were dissolved in DMSO stock solution (10%).
Phosphate buffer (pH 7.0) was used to dilute the DMSO stock solu-
564
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 559 – 565

Tyrosinase Effectors
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The authors thank the French Agence Nationale pour la Recher-
che for financial support (ANR-09-BLAN-0028-01/02/03) and for
grants to M.O., C.B., and C.D. The authors are grateful to the Eu-
ropean COST Program in the framework of which this work was
carried out (COST action CM1003 WG 2).
Keywords: aurone · DFT calculations · enzyme activators ·
enzyme inhibitors · mushroom tyrosinase
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Received: November 14, 2011
Published online on February 3, 2012
ChemBioChem 2012, 13, 559 – 565
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
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