N-(3,5-Dihydroxybenzoyl)-6-hydroxytryptamine as a novel human tyrosinase inhibitor that inactivates the enzyme in cooperation with L-3,4- dihydroxyphenylalanine

Article abstract of DOI:10.1248/cpb.58.1536

N-(3,5-Dihydroxybenzoyl)-6-hydroxytryptamine (2) was a novel inhibitor of L-3,4-dihydroxyphenylalanine (DOPA) oxidase activity of human HMV-II melanoma tyrosinase. The IC₅₀ values for 2 and three reference compounds, N-(3,5-dihydroxybenzoyl)ser

Full text of DOI:10.1248/cpb.58.1536

1536
Note
Chem. Pharm. Bull. 58(11) 1536—1540 (2010)
Vol. 58, No. 11
N-(3,5-Dihydroxybenzoyl)-6-hydroxytryptamine as a Novel Human
Tyrosinase Inhibitor That Inactivates the Enzyme in Cooperation with
L-3,4-Dihydroxyphenylalanine
Yoshimitsu YAMAZAKI* and Yasuhiro KAWANO
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology; 1–1–1 Higashi,
Tsukuba, Ibaraki 305–8566, Japan.
Received July 2, 2010; accepted August 30, 2010; published online September 3, 2010
N-(3,5-Dihydroxybenzoyl)-6-hydroxytryptamine (2) was a novel inhibitor of L-3,4-dihydroxyphenylalanine
(DOPA) oxidase activity of human HMV-II melanoma tyrosinase. The IC50 values for 2 and three reference com-
pounds, N-(3,5-dihydroxybenzoyl)serotonin, 6-hydroxyindole, and kojic acid, were 9.1, 842, 22, and 310 mM, re-
spectively, indicating that the 6-hydroxyindole moiety was more effective than 5-hydroxyindole as the pharma-
cophore of polyphenolic tyrosinase inhibitors and that the inhibitory activity of 6-hydroxyindole was strength-
ened by the link with a resorcinol group. Furthermore, compound 2 exhibited a unique property of inactivating
the human tyrosinase in the presence of low concentrations of DOPA. This inactivation was attenuated by high
concentrations of DOPA and for the most part was irreversible as confirmed by activity stain in native polyacry-
lamide gel electrophoresis and by removal of 2 and DOPA using gel permeation chromatography. Tyrosinase is
the enzyme that oxidizes tyrosine to DOPA and further oxidizes DOPA to the melanin precursor dopaquinone. A
compound such as 2 that inactivates the enzyme in the presence of a small amount of DOPA is therefore attrac-
tive as a new type of tyrosinase inhibitor. Unfortunately, 2 hardly suppressed the melanogenesis in melanoma cell
culture. However, the above strong inhibitory activity and the unique property in the combination with DOPA
suggest that this compound is a useful lead in designing new antimelanogenic agents.
Key words tyrosinase; inhibitor; human; L-3,4-dihydroxyphenylalanine; hydroxyindole; phenolic
Inhibitors of tyrosinase (EC 1.14.18.1) are potentially ap- presence of the substrate DOPA at very low concentrations.
plicable to skin lightening agents.1) A major class of tyrosi-
nase inhibitors is phenolic compounds structurally analogous Results and Discussion
to the substrates, L-tyrosine and L-3,4-dihydroxyphenylala-
The compound 2 was synthesized by coupling of 6-benzyl-
nine (DOPA).1—4) Recently, we found that introduction of a oxytryptamine hemisulfate (10) with 3,5-dihydroxybenzoic
dihydroxyphenyl group into serotonin (that is, a 5-hydroxyin- acid (11) using dicyclohexylcarbodiimide (DCC)7) followed
dole derivative) improved its tyrosinase inhibitory activity.5) by catalytic hydrogenation to remove the benzyl group. Al-
The succeeding study with positional isomers of hydroxyin- though the intrinsic substrate, DOPA, contains an ortho-
doles revealed that 6-hydroxyindole (1) was more potent than diphenol (catechol) group, meta-diphenolic acid 11 was se-
5-hydroxyindole as an inhibitor of human HMV-II melanoma lected because the catechol group was not necessarily supe-
tyrosinase.6) The present study was aimed at evaluating the rior to the meta-diphenol in inhibiting tyrosinase as judged
link of 6-hydroxyindole and a dihydroxyphenyl moiety as the from the facts that 3,5,2Ј,4Ј-tetrahydroxybenzamide was the
design of tyrosinase inhibitors. Thus, the synthesized com- strongest tyrosinase inhibitor among many hydroxy-substi-
pound, N-(3,5-dihydroxybenzoyl)-6-hydroxytryptamine (2), tuted N-benzylbenzamides,8) N-(2,4-dihydroxybenzoyl)sero-
inhibited HMV-II tyrosinase more strongly than 6-hydroxyin- tonin was a stronger inhibitor than N-(3,4-dihydroxyben-
dole (1) and related compounds (Fig. 1) and, moreover, ex- zoyl)serotonin,5) and oxyresveratrol (7) was a much stronger
hibited a unique property of inactivating the enzyme in the inhibitor than piceatannol (8) (Table 1). N-(3,5-Dihydroxy-
benzoyl)serotonin (3) was also synthesized by DCC-medi-
ated coupling of serotonin hydrochloride with 11 for compar-
ison between 6- and 5-hydroxyindole moieties. The in-
hibitory activities of these compounds together with five re-
lated and reference compounds [6-hydroxyindole (1), N-(3,4-
dihydroxybenzoyl)serotonin (4),5) 6-hydroxytryptamine crea-
tinine sulfate salt (5), serotonin hydrochloride (6), and kojic
acid (9)] were tested for the catecholase activity of HMV-II
melanoma tyrosinase. The results are summarized in Table 1.
The IC50 value for compound 2 against HMV-II tyrosinase
was less than one half of the value for 6-hydroxyindole (1),
showing that the introduction of the dihydroxyphenyl moiety
enhanced the inhibitory activity of 1. The compound 2 was
the second strongest inhibitor of HMV-II tyrosinase in Table
1, but the IC50 was only 1.5 mM higher than that for the
strongest inhibitor, oxyresveratrol (7). The IC50 for 2 against
Fig. 1. Chemical Structures of the Compounds
HMV-II tyrosinase was markedly smaller (1/90—1/40) than
∗ To whom correspondence should be addressed. e-mail: y.yamazaki@aist.go.jp
© 2010 Pharmaceutical Society of Japan

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1537
Table 1. Inhibitory Activity of the Compounds for Tyrosinase from
Human HMV-II Melanoma Cellsa)
IC50
(mM)
% inhibition
at 100 mM
Compound
c)
6-Hydroxyindole (1)
22Ϯ1
79Ϯ1
N-(3,5-Dihydroxybenzoyl)-6-
hydroxytryptamine (2)
9.1Ϯ0.3
842Ϯ40
343Ϯ29
12Ϯ1d)
Ͼ1000
7.6Ϯ0.3
Ͼ300
310Ϯ40
94Ϯ1
17Ϯ7
27Ϯ5
77Ϯ4d,e)
5Ϯ3
98
10Ϯ3
27Ϯ1
N-(3,5-Dihydroxybenzoyl)serotonin (3)
N-(3,4-Dihydroxybenzoyl)serotonin (4)
6-Hydroxytryptamine creatinine sulfate (5)b)
Serotonin hydrochloride (6)
Oxyresveratrol (7)
Piceatannol (8)
Kojic acid (9)
Fig. 2. Lineweaver–Burk Plots for DOPA Oxidation by HMV-II Tyrosi-
nase in the Presence of Compound 2
a) Values are meansϮS.D. (standard deviation), nϭ3. b) 5 was dissolved in water
as the sample solution (0.1—10 mM) and water was used as the control. Creatinine
(100 mM) was confirmed to be inactive in this inhibition assay. c) Calculated by sub-
tracting the relative activity (% of control) from 100. d) nϭ2. e) At 30 mM.
Concentrations of 2: 0 (᭺), 1 (᭹), and 3 mM (ᮀ). Every point is the meanϮS.D.
(nϭ3). Plot lines are drawn by least linear regression.
those for the corresponding 5-hydroxyindole derivatives 3
and 4. 6-Hydroxytryptamine creatinine salt (5) also showed a
very small IC50 as compared to serotonin hydrochloride (6).
These facts confirm that the 6-hydroxy group in indole is
more effective than the 5-hydroxy group for the tyrosinase
inhibition. Many kinds of tyrosinase inhibitors have been de-
veloped, but their inhibitory activities were mostly assayed
with mushroom and mouse B16 melanoma tyrosinase.1)
However, the activity of inhibitors against mushroom tyrosi-
nase is often very different from that against mammalian ty-
rosinase,5,6) so if the inhibitor is to be applied to human skin
agents, it is beneficial to test them with human tyrosinase.
Though few data are presently available concerning human
tyrosinase, the IC50 for 2 against HMV-II tyrosinase belongs
to the smallest group among the reported IC50 for arbutin,9,10)
aloesin,10) (ϩ)-imperanene,11) and kinobeon.12) Also in Table
1, the IC50 for 2 is one thirty-fourth that for kojic acid (9),
a standard tyrosinase inhibitor, and as small as that for
oxyresveratrol (7), the active ingredient in the whitening cos-
Fig. 3. HPLC Analysis
Chromatograms for the mixtures of tyrosinase and 10 mM 2 (A, C) or 10 mM DOPA
(B, D) before (A, B) and after incubation at 37 °C for 1 h (C, D).
metics from Morus alba.13) Thus, the inhibitory activity of 2 71% under the same condition (Fig. 3). This result confirms
against the human tyrosinase is higher than or comparable to that 2 is not a substrate of the tyrosinase under this experi-
those of the known inhibitors.
mental condition.
The Lineweaver–Burk plots obtained with different con-
To study the inhibition mechanism in more detail, HMV-II
centrations of 2 for HMV-II tyrosinase activity cross at a tyrosinase was pretreated with compound 2 before assay with
point slightly to the left of the Y-axis, indicating that 2 is a DOPA. The pretreatment mixtures were prepared without or
mixed type inhibitor with respect to DOPA (Fig. 2). The ap- with a small amount of DOPA to investigate the effect of 2
parent Michaelis constant (Km) was 1.1 mM and replots of the on the enzyme turning over in catalysis. Thus, HMV-II ty-
slope and Y-intercept of the lines in Fig. 2 vs. concentration rosinase solutions containing 2 and/or DOPA, or none of
of 2 gave the inhibitor constants Kiϭ1.7 mM for Enzyme- them were incubated at 37 °C for 1 h and then DOPA was
Inhibitor complex and KiЈϭ53 mM for Enzyme-Substrate- added to the solutions to 2.5 mM (i.e., the concentration used
Inhibitor complex, respectively.14) Although the simple in the standard assay). After standing at room temperature
Michaelis–Menten kinetics is not properly applied here be- for 24 h, the mixtures pretreated with 2 plus 10 mM DOPA
cause of the enzyme inactivation induced by 2 and DOPA showed melanin formation as low as the control level (autox-
(see the later section), it is most likely that compound 2 pri- idation of DOPA), while all mixtures pretreated with 2 alone
marily competes for the enzyme active site with DOPA. Ty- became dark with much melanin (Fig. 4A). This pretreatment
rosinase contains a binuclear copper complex in the active effect of an inhibitor plus DOPA was not found with com-
center for the substrate binding and oxidation. Since the two pound 3 though its concentration was ten times that used for
resorcinol derivatives 2 and 3 showed the very different in- 2. In addition, 6-hydroxyindole (1) and oxyresveratrol (7)
hibitory activities, 2 is bound to the copper center for the in- showed a similar combination effect with DOPA on the
hibition probably through 6-hydroxyindole moiety rather melanin suppression (data not shown). The tyrosinase activ-
than the resorcinol moiety. In addition, HPLC analysis ity itself decreased by the pretreatment with 2 plus DOPA
showed that the peak of 2 did not change after incubation and the activity loss increased with the preincubation time.
with HMV-II tyrosinase for 1 h, while DOPA diminished by When treated only with 2 or DOPA, the tyrosinase activity

1538
Vol. 58, No. 11
Fig. 4. Tyrosinase Inactivation by 2 in the Presence of Low Concentrations of DOPA
(A) Melanin formation by tyrosinase preincubated with 0—10 mM 2 or 0—100 mM 3 without or with 10 mM DOPA for 1 h. After the preincubation, DOPA was added to 2.5 mM
and then the plate was left at room temperature for 24 h. Control 1 contained 50 mM phosphate buffer (pH 6.8, without tyrosinase and the inhibitors) but received 2.5 mM DOPA.
Control 2 contained and received the buffer only. The wells of preincubation with 5 or 10 mM 2 plus 10 mM DOPA are as light as the well of control 1, while those of preincubation
with 2 alone or with 3 and/or DOPA are all dark, indicating that the simultaneous pretreatment with 2 and DOPA suppressed the melanin formation and that 3 was not effective in
suppressing the melanin even with DOPA. (B) Effect of the length of preincubation time. Samples contained tyrosinase and 10 mM 2 plus 10 mM DOPA (᭹), 10 mM 2 alone (᭡),
10 mM DOPA alone (ᮀ), or none of them (᭺). The preincubation time was 0, 3, 10, 30, and 60 min. (C) Effect of the concentrations of DOPA and 2 in the preincubation for 10 min.
Samples contained tyrosinase, 2 [0 (᭺), 1 (᭡), 3 (᭿), or 10 mM (᭹)], and DOPA (0, 1, 10, 100, 1000, or 2500 mM). The vertical axis in B and C represents the residual enzyme ac-
tivity expressed as % of control (without 2 and DOPA at time 0). Every point is the meanϮS.D. (nϭ3). (D) Tyrosinase activity staining after native PAGE for samples preincubated
without or with 2 and/or DOPA. Their concentrations (mM) are indicated on the top. M indicates molecular weight marker proteins (kDa). (E) Coomassie brilliant blue staining of
the same gel as above. Bands of 59 or 51 kDa serve as protein loading reference. (F) Western blot of tyrosinase electrophoresed under the same condition as above. Color prints of
D and E, see the Graphical Abstract.
slightly changed in the experimental period (Fig. 4B). The after passing through a Sephadex® G-50 column (Fig. 5B).
loss of tyrosinase activity was dependent on the dose of 2 The initial velocity (% of the control) for the sample pre-
and DOPA (Fig. 4C). The plots of the residual activities de- treated only with 2 recovered from 42 to 78% by the GPC
termined after preincubation for 10 min show that DOPA it- treatment (cf. plots c and cЈ), but that for the sample pre-
self caused the decrease in tyrosinase activity at the high treated with 2 plus DOPA recovered from 0% (plot d) to only
concentrations, but that the effect of DOPA was increased by 9% (plot dЈ). This enzyme activity did not significantly in-
the presence of 2. The maximum effect of DOPA was found crease when the same sample (the column eluate) was left at
at about 100 mM in the combination with 10 mM 2. The entity room temperature for 6 h before initiating the assay (plot dЉ).
of tyrosinase treated with 2 and DOPA was investigated by
The most interesting property of compound 2 is that the
the method of activity stain in native polyacrylamide gel inhibition of HMV-II tyrosinase by 2 is greatly strengthened
electrophoresis (PAGE).15) The untreated tyrosinase gave the by the presence of low concentrations of DOPA. Since this
main activity band (colored red) at molecular weight of inhibition remains long after the condition is altered by the
67 kDa (lit.16) 62610 Da), whose migration was consistent addition of high concentrations of DOPA, it might be reason-
with that of the immunologically stained tyrosinase band able to conclude that the enzyme is inactivated by the treat-
(Figs. 4D, F; see also the color print in Graphical Abstract). ment with 2 plus DOPA. The disappearance of activity stain
The activity band for the samples treated with 10 mM 2 plus in PAGE (Fig. 4D) and the limited recovery of activity after
10 or 2500 mM DOPA was faint and it did not appear for the GPC separation (Fig. 5B) imply that this inactivation is
samples treated with 10 mM 2 plus 100 or 1000 mM DOPA. mostly irreversible. Tyrosinase is known to be inactivated
The activity band was also lighter for the samples treated during the oxidation of DOPA17,18) and several phenolic sub-
with 1000 or 2500 mM DOPA alone than those for the sam- strates.19—21) This type of inactivation is called suicide inacti-
ples treated with 0—100 mM DOPA. This coloration is nearly vation, whose mechanism has been discussed for many
parallel to the enzyme activity shown in Fig. 4C, suggesting years.17—23) For example, a two-electron transfer from the
that the activity decrease by 2 and DOPA is due to disappear- substrate to the active center Cu2ϩ ion leading to its libera-
ance of the main tyrosinase activity.
tion as a Cu0 atom was proposed as the mechanism for inacti-
The combination effect of 2 with DOPA was further stud- vation of tyrosinase in the enzymatic oxidation of
ied by separating the pretreated enzyme from 2 and DOPA by resorcinol.21) Compound 2 has a resorcinol moiety, but this is
gel permeation chromatography (GPC). Samples containing probably not the cause of the present inactivation, since an-
tyrosinase without or with 10 mM 2 and/or 10 mM DOPA were other resorcinol derivative 3 did not suppress the melanin
incubated at 37 °C for 1 h and then directly assayed with formation (Fig. 4A). The above-mentioned electron transfer
2.5 mM DOPA (Fig. 5A) or assayed with the protein fractions might be possible from the 6-hydroxyindole moiety with pro-

November 2010
1539
Fig. 5. Tyrosinase Activity Pretreated with 2 and/or DOPA and That Recovered from the Mixtures by GPC
(A) Monitor of DOPA oxidation by tyrosinase 1 h preincubated with 10 mM 2 and 10 mM DOPA (plot d), 10 mM 2 alone (c), 10 mM DOPA alone (b), or none of them (a). (B) Moni-
tor of DOPA oxidation by tyrosinase in the protein fractions from GPC of the same preincubated samples as above. Plot aЈ, bЈ, cЈ, dЈ, and dЉ correspond to plot a, b, c, d, and d, re-
spectively. Plot dЉ was recorded after the column eluate was left at room temperature for 6 h. The total activities indicated by plot a and plot aЈ were almost equal as calculated with
the volumes of assay mixture and sample solution.
duction of a conjugated iminoketone, but presently we have and further oxidizes DOPA to the melanin precursor
no evidence for it. Although DOPA may act as the suicide dopaquinone. A compound such as 2 that inactivates the en-
substrate in the present phenomenon, its effective concentra- zyme in the presence of a small amount of DOPA is therefore
tion (around 100 mM) is considerably low as compared to attractive as a new type of tyrosinase inhibitor. Unfortu-
those (0.25—10 mM) used for suicide inactivation under aer- nately, compound 2 was rather inactive in suppressing the
obic conditions.17,18) The effectiveness of DOPA at such a melanogenesis in cultured HMV-II cells. The melanin forma-
low concentration implies a possibility that 2 enhances the tion in these cells was not significantly suppressed by 3—
suicide inactivation by DOPA, for instance, through confor- 20 mM 2, while 10—30 mM 2 decreased the cell viability (data
mational change of the enzyme protein by binding. However, not shown). These problems must be solved by further modi-
this mechanism does not explain the fact that there was the fication of the chemical structure. However, the above strong
optimum concentration for DOPA in the combination effect inhibitory activity and the unique property in the combina-
with 2 (Fig. 4C). One possible interpretation of this phenom- tion with DOPA suggest that this compound is a useful lead
enon is that a specific binding of 2 to oxy- or red-tyrosinase in designing new antimelanogenic agents. The mechanism of
might result in the inactivation of the enzyme. The copper the present inactivation is to be studied through detailed ki-
complex in tyrosinase is cycled among met-, oxy-, and red- netic analysis using purified HMV-II tyrosinase in future.
(or deoxy-)state in the catalytic process.1) The oxy- or red-ty-
Experimental
rosinase must be generated in the enzymatic reaction with
Materials 6-Hydroxytryptamine creatinine sulfate (5) and 2-(6-benzyl-
DOPA, since the native (resting) tyrosinase is mostly in the
met-state.23) The proportion of the oxy- or red-tyrosinase is
established by the relative concentrations of DOPA, inhibitor,
O2, and the enzyme. If DOPA is in excess over 2, the enzyme
proceeds to the normal reaction step to avoid complexing
with 2. This would explain the need for the limited concen-
tration of DOPA for the present inactivation by 2. On the
other hand, the suicide inactivation by DOPA should be irre-
versible as expected from the zero-valent copper formation
mechanism.19,21) The GPC experiment indicated that the re-
moval of 2 and DOPA from the pretreated enzyme restored
only 9% of the control activity and this recovery increased no
further during the 6-h standing (Fig. 5B). This fact suggests
that the major part of HMV-II tyrosinase is irreversibly inac-
tivated by the treatment with 2 plus DOPA, while the minor
part is reversibly inactivated. The coexistence of two kinds of
inactivation might be due to a heterogeneity of the tyrosi-
nase. Now, it is not clear whether 2 causes the present inacti-
vation by its permanent binding to the enzyme, or 2 merely
promotes the DOPA-induced suicide inactivation, or 2 itself
serves as a suicide substrate. Some irreversible inhibitors
were reported for mushroom tyrosinase,24,25) but few are
known for human tyrosinase. Furthermore, the combination
effect of inhibitors with DOPA has not been reported for any
tyrosinase.
oxyindolyl)ethylamine hemisulfate (10) were purchased from Toronto Re-
search Chemicals Inc. (North York, ON, Canada), 6-hydroxyindole (1),
piceatannol (8), and serotonin hydrochloride (6) were from Wako Pure
Chemicals Inc. (Osaka, Japan), kojic acid (9) and 3,5-dihydroxybenzoic
acid (11) were from Tokyo Chemical Industries Ltd. (Tokyo, Japan), and
oxyresveratrol (7) was from Hangzhou Great-Forest Biomedical Ltd.
(Hangzhou, P. R. China).
Synthesis N-(3,5-Dihydroxybenzoyl)-6-hydroxytryptamine (2) was syn-
thesized by the DCC method.7) The amine salt 10 (100 mg) was suspended
in 5 ml dimethylformamide (DMF) containing 2 ml pyridine and mixed with
a solution of 100 mg 3,5-dihydroxybenzoic acid (11) and 100 mg DCC in
0.5 ml DMF. The mixture was stirred at room temperature for 1 h. The insol-
uble material was collected by centrifugation, suspended in 1 ml dimethyl-
sulfoxide containing 0.2 ml pyridine, and supplemented with 200 mg 11 and
200 mg DCC. To the supernatant from the above centrifugation, 200 mg 11
and 400 mg DCC were added. After stirring at room temperature overnight,
the two mixtures were filtered and the filtrates were concentrated by a rotary
evaporator. The residues were respectively dissolved in 30 ml ethyl acetate,
and washed successively with 10% citric acid, 10% NaHCO3, and saturated
NaCl in water. Finally, the ethyl acetate solutions were combined, dried over
Na2SO4, concentrated by an evaporator, and applied to a silica gel column.
The eluate with ethyl acetate was dissolved in 4 ml ethanol containing 0.2 ml
acetic acid, and hydrogenated over 30 mg Pd/C at an atmospheric pressure
for 4 h. The catalyst was filtered off and the filtrate was concentrated to give
a residue, which was purified by silica gel column chromatography. The
main product (2) was eluted with ethyl acetate and crystallized from ethyl
acetate and benzene as fine needles (34 mg, 34% yield), dec. 213—217 °C,
Anal. Calcd for C17H16N2O4·1/2H2O: C, 63.54; H, 5.33; N, 8.72%. Found: C,
63.37; H, 5.31; N, 8.46%; FAB-MS m/z 313.1197 ([MϩH]ϩ) (Calcd
1
C17H17N2O4ϭ313.1187); and H-NMR (270 MHz, acetone-d6) d: 2.99 (2H,
Tyrosinase is the enzyme that oxidizes tyrosine to DOPA t, Jϭ7 Hz, CH2), 3.64 (2H, m, CH2N), 6.47 (1H, t, Jϭ2 Hz, H-4), 6.64 (1H,

1540
Vol. 58, No. 11
dd, Jϭ8 and 2 Hz, H-5Ј), 6.82 (1H, d, Jϭ2 Hz, H-7Ј), 6.84 (2H, d, Jϭ2 Hz, horseradish peroxidase rabbit anti-goat IgG (HϩL) conjugate (Zymed Labo-
H-2,5), 6.99 (1H, m, H-2Ј), 7.43 (1H, d, Jϭ8 Hz, H-4Ј), 7.56 (1H, br s,
NHCO), 7.82 (1H, br s, OH), 8.41 (2H, br s, OHϫ2), and 9.64 (1H, br s, H-
1Ј).
ratories Inc., San Francisco, CA, U.S.A.).
Inhibitor Removal by Gel Permeation Chromatography Samples
(1 ml each) containing 2.7 mU/ml tyrosinase, 0.05% NP-40 (derived from
the enzyme extract), and 10 mM 2 plus 10 mM DOPA, 10 mM 2 alone, 10 mM
DOPA alone, or none of them in 47 mM phosphate buffer (pH 6.8) were pre-
pared and incubated at 37 °C for 1 h. Then, 12.5 mM DOPA in 50 mM phos-
N-(3,5-Dihydroxybenzoyl)serotonin (3) was similarly prepared from 6 and
11, crystalline solid, 94 mg (64% yield), mp 113—117 °C and dec. 215—
218 °C (with multiple phase transitions), FAB-MS m/z 313 ([MϩH]ϩ)
1
(Calcd C17H16N2O4ϭ312). H-NMR (270 MHz, acetone-d6) d: 2.98 (2H, t, phate buffer (pH 6.8) (0.25 ml each) was added to the pretreated mixtures
Jϭ7 Hz, CH2), 3.66 (2H, m, CH2N), 6.51 (1H, t, Jϭ2 Hz, H-4), 6.71 (1H,
dd, Jϭ8 and 2 Hz, H-6Ј), 6.84 (2H, d, Jϭ2 Hz, H-2,5), 7.06 (1H, d, Jϭ2 Hz, hand, the same samples as above were incubated at 37 °C for 1 h, and then
H-4Ј), 7.10 (1H, br s, H-2Ј), 7.46 (1H, d, Jϭ8 Hz, H-7Ј), 7.72 (1H, br s,
applied to a Sephadex® G-50 column (10 cm3) equilibrated with 50 mM
and absorbance at 475 nm was continuously recorded at 24 °C. On the other
NHCO), 7.98 (1H, br s, OH), 8.33 (1H, br s, OH), and 9.75 (1H, br s, H-1Ј). phosphate buffer (pH 6.8) containing 0.05% NP-40. Elution was performed
N-(3,4-Dihydroxybenzoyl)serotonin (4) was synthesized in the previous with the same buffer and the eluate was divided into 1-ml fractions. Proteins
work.5)
were eluted in Fr. 4 and 5, while 2 and DOPA were eluted in Fr. 9—16 as ev-
Tyrosinase Assay Human HMV-II melanoma cells (Dai-Nippon-Sumi- idenced by UV monitoring at 280 nm. From the combined solution of Fr. 4
tomo Pharmaceuticals Inc., Osaka, Japan) were grown in RPMI-1640 and 5, aliquots (0.8 ml each) were taken and mixed with 0.2 ml 12.5 mM
medium containing 15% fetal bovine serum. Tyrosinase was extracted from DOPA to assay tyrosinase activity. The same experiment was repeated with
cells with a lysis buffer containing 150 mM NaCl, 1 mM ethylenediaminete- the sample for 2 plus DOPA, but the eluate from the column was left at room
traacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1% polyoxyethylene(9) temperature for 6 h before initiating the assay as above.
octylphenyl ether (NP-40), 0.1% Na dodecylsulfate, and 0.1% Na deoxy-
cholate in 10 mM Tris/HCl buffer (pH 7.4) by sonication at 4 °C. After re-
moving the cell debris by centrifugation (15000 rpm, 10 min), the super-
natant was used as the tyrosinase source. Protein was determined by the
bicinchoninic acid method with bovine serum albumin as standard. The ty-
rosinase activity was assayed with DOPA as the substrate.5) Briefly, assay so-
lutions were prepared by mixing 0.94 ml 50 mM phosphate buffer (pH 6.8)
containing 2.6 mM DOPA and 10 ml ethanol containing 0—30 mM test com-
pound, unless otherwise stated. The reaction was started by adding 50 ml en-
zyme solutions adequately diluted with the lysis buffer to the substrate solu-
tions prewarmed to 37 °C, and the dopaquinone formation during incubation
for 5 min was determined by light absorption at 475 nm (eϭ3700 MϪ1 cmϪ1).
Usually, 2.5—3 mU enzyme5) was applied per assay.
HPLC Analysis Samples containing 2.5 mU/ml tyrosinase, 10 mM 2 or
10 mM DOPA, and 10 mM adenine (as an internal standard) in 50 mM phos-
phate buffer (pH 6.8) were prepared and 0.45 ml of each sample was mixed
with 50 ml 3% trifluoroacetic acid (TFA) immediately or after incubation at
37 °C for 1 h. After removing the precipitate by centrifugation, the solutions
were subjected to HPLC with a Capcell Pak C18 column, 4.6ϫ150 mm (Shi-
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