The metabolic fate of ortho-quinones derived from catecholamine metabolites

Article abstract of DOI:10.3390/ijms17020164

ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by enzymes such as tyrosinase or by transition metalions. Neuromelanin, a dark pigment present in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid (DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible spectrophotometry. HPLC analysis after reduction with NaBH4 or ascorbic acid enabled measurement of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric examination showed that the ortho-quinones initially formed underwent extensive degradation at pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from DOPE-quinone was DOPEG which was produced through the addition of a water molecule to the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc) and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min. This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are converted to quinone methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The unstable quinone methides afford stable alcoholic or carbonyl products.

Full text of DOI:10.3390/ijms17020164

International Journal of
Molecular Sciences
Article
The Metabolic Fate of ortho-Quinones Derived from
Catecholamine Metabolites
Shosuke Ito 1, Yuta Yamanaka 1, Makoto Ojika 2 and Kazumasa Wakamatsu 1,
*
1
Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi 470-1192,
Japan; sito@fujita-hu.ac.jp (S.I.); fuh-tech.mau-in.yu.0831@qc.commufa.jp (Y.Y.)
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Nagoya, Aichi 464-8601, Japan; ojika@agr.nagoya-u.ac.jp
2
*
Correspondence: kwaka@fujita-hu.ac.jp; Tel.: +81-562-93-2518; Fax: +81-562-93-4595
Academic Editor: Manickam Sugumaran
Received: 20 November 2015; Accepted: 22 January 2016; Published: 27 January 2016
Abstract: ortho-Quinones are produced in vivo through the oxidation of catecholic substrates by
enzymes such as tyrosinase or by transition metal ions. Neuromelanin, a dark pigment present
in the substantia nigra and locus coeruleus of the brain, is produced from dopamine (DA) and
norepinephrine (NE) via an interaction with cysteine, but it also incorporates their alcoholic and
acidic metabolites. In this study we examined the metabolic fate of ortho-quinones derived from
the catecholamine metabolites, 3,4-dihydroxyphenylethanol (DOPE), 3,4-dihydroxyphenylethylene
glycol (DOPEG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylmandelic acid
(DOMA). The oxidation of catecholic substrates by mushroom tyrosinase was followed by UV-visible
spectrophotometry. HPLC analysis after reduction with NaBH4 or ascorbic acid enabled measurement
of the half-lives of ortho-quinones and the identification of their reaction products. Spectrophotometric
examination showed that the ortho-quinones initially formed underwent extensive degradation at
pH 6.8. HPLC analysis showed that DOPE-quinone and DOPEG-quinone degraded with half-lives
of 15 and 30 min at pH 6.8, respectively, and >100 min at pH 5.3. The major product from
DOPE-quinone was DOPEG which was produced through the addition of a water molecule to
the quinone methide intermediate. DOPEG-quinone yielded a ketone, 2-oxo-DOPE, through the
quinone methide intermediate. DOPAC-quinone and DOMA-quinone degraded immediately with
decarboxylation of the ortho-quinone intermediates to form 3,4-dihydroxybenzylalcohol (DHBAlc)
and 3,4-dihydroxybenzaldehyde (DHBAld), respectively. DHBAlc-quinone was converted to
DHBAld with a half-life of 9 min, while DHBAld-quinone degraded rapidly with a half-life of 3 min
This study confirmed the fact that ortho-quinones from DOPE, DOPEG, DOPAC and DOMA are
converted to quinone methide tautomers as common intermediates, through proton rearrangement
or decarboxylation. The unstable quinone methides afford stable alcoholic or carbonyl products.
.
Keywords: catechols; ortho-quinones; quinone methide; catecholamines; neuromelanin
1. Introduction
Neuromelanin is a complex polymeric compound present in the human central nervous system
which is synthesized mainly in dopaminergic neurons of the substantia nigra and in noradrenergic
neurons of the locus coeruleus [
arises during brain aging and there is evidence that neuromelanin is involved in the pathogenesis
of neurodegenerative diseases such as Parkinson’s disease [ ]. Notably, the substantia nigra and
1,2]. The synthesis and accumulation of neuromelanin inside neurons
3–5
locus coeruleus are the areas in the brain with the highest concentration of neuromelanin pigment [6].
The biosynthesis and structure of neuromelanin remains poorly understood, mainly because of
difficulties in its isolation and the complexity of its structure [
1
,2]. Nevertheless, our degradative
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Int. J. Mol. Sci. 2016, 17, 164
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approach suggested that the pigmented part of substantia nigra neuromelanin is derived from
dopamine (DA) and cysteine in a molar ratio of 2:1 [ ]. In addition, it was recently suggested
that various catecholic metabolites are incorporated into neuromelanin from the substantia nigra and
locus coeruleus, including 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylethanol (DOPE, 1)
3,4-dihydroxyphenylethylene glycol (DOPEG, ), 3,4-dihydroxyphenylacetic acid (DOPAC, ) and
3,4-dihydroxymandelic acid (DOMA, ), which are metabolites of dopamine (DA) and norepinephrine
7,8
,
2
3
4
(NE) formed by the oxidative deamination by monoamine oxidase followed by reduction/oxidation
(Figure 1) [9–11].
Oxidation of those catecholamine metabolites leads to the production of ortho-quinones, although
the nature of the oxidants remains controversial [12]. Once formed, the ortho-quinones are trapped
rapidly by cysteine (or other thiols) as long as it is present [13]. The cysteine adducts are oxidized
to give rise to pheomelanic pigments [
ortho-quinones derived from DA, NE and DOPA are cyclized to give aminochromes and eventually
eumelanic pigments [ 10 13]. Thus, the production of neuromelanin is considered as a detoxifying
mechanism of otherwise toxic ortho-quinones [14]. The fate of ortho-quinones derived from the
catecholamine metabolites DOPE ( ), DOPEG ( ), DOPAC ( ) and DOMA ( ) are studied by kinetic
and spectroscopic techniques and the results are presented in this paper.
9,10]. After consumption of available cysteine in neurons,
9, ,
1
2
3
4
Figure 1. Possible participation of catecholic metabolites known to be present in various regions of the brain
that may be incorporated into neuromelanin [ ]. In addition to dopamine (DA) and norepinephrine
(NE) and the corresponding Cys-derivatives, these other metabolites are also thought to be incorporated
into neuromelanin [ 10]. substantia nigra, SN; locus coeruleus, LC; 3,4-dihydroxyphenylethanol,
7,8
9,
DOPE; 3,4-dihydroxyphenylethylene glycol, DOPEG; 3,4-dihydroxyphenylacetic acid, DOPAC;
3,4-dihydroxyphenylmandelic acid, DOMA; 3,4-dihydroxyphenylalanine, DOPA; cysteine, cys. The
(O) represents the oxidants. Adapted from [10] with minor modifications.
In this study, we compared the reactivities of those ortho-quinones regarding their half-lives and
degradation products. ortho-Quinones are produced by oxidation with mushroom tyrosinase. The
fates of ortho-quinones were examined by two methods [15,16]: UV-visible spectrophotometry to detect
ortho-quinones with an absorption maximum around 400 nm and HPLC to follow the degradation
of ortho-quinones and the appearance of products after reduction to the corresponding catechols
by NaBH4 or ascorbic acid. The results suggest that ortho-quinone products from catecholamine
metabolites may not be incorporated into neuromelanin, but exert neurotoxicity as such because
ortho-quinones are highly reactive compounds that lead to cytotoxicity through their binding to thiol
enzymes and the production of reactive oxygen species [17–19].

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2. Results
2.1. Spectrophotometric Examinations
The UV-visible spectrophotometric examinations were performed at a 100
µ
M concentration of
the four catecholamine metabolites in 50 mM sodium phosphate buffer, pH 6.8 or 5.3, at 37 ˝C. The
oxidation was initiated by adding 50 U/mL mushroom tyrosinase and was then periodically followed.
Spectral changes at pH 6.8 are shown in Figure 2. DOPE (
the corresponding ortho-quinones (400 nm) in 2 min and were then gradually degraded to ill-defined
products after 60 min (Figure 2A,B). DOPAC ( ) was converted to an ortho-quinone (400 nm) in 2 min
but was rapidly degraded with a broader absorption (Figure 2C). DOMA ( ) was oxidized to an
ortho-quinone having an absorption maximum at 420 nm which may correspond to the ortho-quinone
oxidation product of 3,4-dihydroxybenzaldehyde (DHBAld ( ); Figure 2C,D; see below for the
1) and DOPEG (2) were oxidized to
3
4
7
identification). Spectral changes at pH 5.3 are shown in Figure S1. At pH 5.3, the ortho-quinones
formed were much more stable than at pH 6.8 and showed more subtle spectral changes in 60 min.
Figure 2. Time course of the degradation of ortho-quinone products from catecholamine metabolites,
(A) DOPE (1); (B) DOPEG (2); (C) DOPAC (3); (D) DOMA (4). UV-visible spectral changes associated
with the tyrosinase catalyzed oxidation of different catechols at pH 6.8. Each experiment was performed
twice with similar results.
2.2. HPLC Examinations
HPLC examinations were performed for the products after reduction of ortho-quinones with
NaBH4 or ascorbic acid to the corresponding catechols (Figure 3 and Figure S2). The difference between
reduction with NaBH4 and with ascorbic acid is that NaBH4 reduces not only the ortho-quinone moiety
but also the carbonyl group to a hydroxyl group, whereas ascorbic acid is able to reduce only the
former. HPLC conditions for the product analysis are summarized in Table 1.
Table 1. Half-lives of ortho-quinone products from catecholamine metabolites (1–4) and HPLC
conditions used to follow the oxidation.
Half-Life (t1/2, min)
HPLC Conditions 1
Ortho-Quinone Derived from
with NaBH4
with Ascorbic Acid
pH 6.8
pH 5.3
pH 6.8
pH 5.3
DOPE (1)
DOPEG (2)
14.6
30.9
9.1
128
187
15.2
30.0
9.5
114
154
80:20, 35 ˝
90:10, 35 ˝
85:15, 50 ˝
85:15, 50 ˝
C
C
C
C
DHBAlc (6) from DOPAC (3)
DHBAld (7) from DOMA (4)
1
46.8
47.8
3.0 2
3.3 2
3.0 2
3.3 2
2
The mobile phase of 0.4 M formic acid:methanol, v/v (%), and the column temperature; These values
were subject to experimental errors because of the rapid degradation of DHBAld (
3,4-dihydroxybenzylalcohol; DHBAld, 3,4-dihydroxybenzaldehyde.
7)—quinone. DHBAlc,

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HPLC following the oxidation of DOPE (1) at pH 6.8 showed a gradual degradation of DOPE
(
1
)-quinone (analyzed as DOPE (
of a more hydrophilic compound with a shorter retention time compared to DOPE (
The compound was found to have a UV absorption spectrum almost identical to DOPE (
1
) after reduction with ascorbic acid (AA) or NaBH4) with production
) (Figure 3A,B).
) but different
1
1
from the 3,4,6-trihydroxy chromophore of 6-hydroxydopamine (Figure S3A). It was then identified as
DOPEG (2) by co-injection on HPLC.
Figure 3. HPLC following the degradation of ortho-quinone products from catecholamine metabolites
at pH 6.8. (
A
,B
) DOPE (
1); (
C,
D) DOPEG (
2
); (
E,
F) DOPAC (
3
); (
G
,H) DOMA (
4). For A, C, E and G,
the oxidation was stopped by the addition of NaBH4; For
B,
D,
F
and , the oxidation was stopped
H
by the addition of ascorbic acid (AA). For the sake of clarity, the yields of DOPEG ketone (5) were
multiplied by a factor of 10 in (D) and those of DHBAld (7) were by a factor of 5 in (F).
When analyzed after ascorbic acid reduction, the oxidation of DOPEG (
longer retention time than DOPEG (Figure 3D). The compound was found to have a UV absorption
spectrum almost identical to DHBAld ( ) (Figure S3B), suggesting it to have a carbonyl group adjacent
to the 3,4-dihydroxyphenyl ring. To confirm its structure, the compound was prepared by NaIO4
oxidation of DOPEG ( ) and isolated, although in a low yield (1%), by preparative HPLC. The
compound was reduced by NaBH4 to afford DOPEG ( ) (Figure S4), suggesting that it is 2-oxo-DOPE
). This identification was then confirmed by direct comparison with an authentic sample (see below).
When analyzed after NaBH4 reduction, the 2-oxo-DOPE ( ) actually formed was detected as DOPEG
) (Figure 3C). Although the yield of 2-oxo-DOPE ( ) is very low, it is a major product in the early
phase of the degradation of DOPEG ( )-quinone. Its low yield can be ascribed to the slow conversion
from DOPEG ( )-quinone and the high reactivity of its quinone form, similar to the unstable DHBAld
)-quinone (see below). It should also be noted that ortho-quinones of DOPE ( ) and DOPEG ( ) did
2) gave a product with a
7
2
2
(5
5
(2
5
2
2
(7
1
2
not undergo a cyclization reaction to form a five-membered ring. This is in contrast to the formation of
a six-membered ring from the ortho-quinone of rhododendrol through the hydroxy group [13].
When analyzed after ascorbic acid reduction, the oxidation of DOPAC (
3,4-dihydroxybenzylalcohol (DHBAlc; ) as an initial and major product, which was gradually
converted to DHBAld ( ), although in low yield (
and DHBAld ( ) was confirmed by co-injection with authentic samples. The oxidation of DOMA (
yielded DHBAld (
polymeric product (Figures 2D and 3H). The oxidation of DHBAld (
3) yielded
6
7
7
; Figure 3F). The identification of DHBAlc (
6
4
)
)
7
7
) as an initial and major product, which rapidly disappeared to give ill-defined
) itself afforded similar results
7

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on spectrophotometric and HPLC examinations (data not shown). The 3,4-dihydroxybenzoic acid, a
possible product, was not detected. When analyzed after NaBH4 reduction, the DHBAld (7) actually
formed was detected as DHBAlc (6) (Figure 3E,G).
In summary, 2-oxo-DOPE (
ESI(+)/MS, and by co-injection on HPLC with the authentic sample provided by Manickam Sugumaran
5) was identified as the product of DOPEG (2
)-quinone by 1H-NMR,
(Figures S5 and S6). The catechol products from DOPE (1)-quinone, DOPAC (3)-quinone, and DOMA
(4)-quinone were identified with the authentic samples.
HPLC following oxidation was also performed at pH 5.3 to examine the effect of pH on the fate of
ortho-quinones as DOPEG ( ), DHBAlc ( ) and DHBAld ( ) by comparison. As shown in Figure S2, the
2
6
7
ortho-quinones (analyzed as catechols after reduction) were much more stable at pH 5.3 than at pH 6.8,
except for DHBAld (7) which degraded at similar rates at both pHs.
The half-lives of the ortho-quinones produced in this study are summarized in Table 1. The
reduction with NaBH4 or ascorbic acid gave similar results with differences within 10% of the averages.
The stability of ortho-quinones was in the order of DOPEG (
2)-quinone > DOPE (1)-quinone > DHBAlc
(6
)-quinone > DHBAld ( )-quinone. These quinones were more reactive at pH 6.8 by a factor of 5–8 as
7
compared to pH 5.3. An exception is that DHBAld (7)-quinone was as reactive at pH 5.3 as at pH 6.8.
3. Discussion
The catecholamines DA and NE are converted to alcoholic and acidic metabolites DOPE (
DOPEG ( ), DOPAC ( ) and DOMA ( ) in neurons (Figure 1). Our recent studies provided evidence
for the incorporation of those catecholic metabolites into neuromelanins in the substantia nigra and the
locus coeruleus in a pheomelanic structure [ 10]. This result implies the involvement of ortho-quinone
intermediates that are trapped by cysteine to form cysteinyl derivatives [ 10 13]. In the present
study, we used mushroom tyrosinase as an oxidant because it specifically and mildly oxidizes only
catecholic substrates (and phenolic substrates) [15 16 20]. Our results show that the quinone methide
intermediates are produced from the ortho-quinones of through deprotonation in DOPE ( ) and
DOPEG ( ) or through decarboxylation in DOPAC ( ) and DOMA ( ). Those quinone methides
1),
2
3
4
9,
9
, ,
,
,
1
–
4
1
2
3
4
undergo the addition of a water molecule to form alcohols or tautomerization to form carbonyl
compounds. These reaction pathways are summarized in Figure 4.
Figure 4. The metabolic fate of ortho-quinone products from catecholamine metabolites DOPE (
1),
DOPEG (
NaBH4 or ascorbic acid (AA) to afford DOPEG (
addition of AA to afford 2-oxo-DOPE (
AA to afford DHBAlc ( ) and DHBAld (
AA to afford DHBAld (7).
2
), DOPAC (
3
) and DOMA (
4
). The oxidation of DOPE (
1
) was stopped by the addition of
2
). The oxidation of DOPEG (2) was stopped by the
5
). The oxidation of DOPAC ( ) was stopped by the addition of
3
6
7
). The oxidation of DOMA (4) was stopped by the addition of

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The metabolic fate of DOPE (1)-quinone was first reported by Sugumaran et al. [21] using a
preparation of cuticular enzyme(s) from Sarcophaga bullota. With that insect enzyme(s) they were
able to show a rapid conversion of DOPE ( )-quinone to DOPEG ( ) through the quinone methide
intermediate. DOPEG ( ) was then gradually converted to 2-oxo-DOPE ( ). However, that series
of reactions did not proceed beyond the DOPE ( )-quinone stage when DOPE ( ) was oxidized by
1
2
2
5
1
1
mushroom tyrosinase [21]. The authors concluded that with the cuticular enzyme(s), quinone methide
tautomerase acts on the ortho-quinone intermediate(s) to form quinone methide(s). The discrepancy
between our results and theirs could be attributed to the fact that they followed the oxidation of DOPE
(
1
) at a low concentration of tyrosinase while we followed the fate of DOPE (
produced rapidly (<2 min) by a high concentration of tyrosinase. Later studies on the fate of DOPE
)-quinone by d’Ischia’s group reported the production of a number of products, including dimers,
but not DOPEG ( ) [22 23]. The discrepancy may arise from differences in the oxidant (tyrosinase vs.
1)-quinone that was
(
1
2
,
peroxidase plus hydrogen peroxide), the concentration (0.1 vs. 1 mM) and/or the pH (6.8 vs. 7.4). The
concentration is exceptionally important because the quinone methide formation is a first-order reaction
that is independent of the concentration, whereas the dimer production is a concentration-dependent,
second-order reaction.
The metabolic fate of DOPEG (
above-mentioned report by Sugumaran et al. [21]. In the present study, we identified the ketone
metabolite of DOPEG ( ), 2-oxo-DOPE ( ), in the oxidation mixture when the oxidation was stopped
2)-quinone has not been extensively studied except for the
2
5
by ascorbic acid (but not by NaBH4). The yield of this ketone was very low because of its high reactivity
in the reaction mixture. In this connection, it should be mentioned that a similar, high reactivity of NE
ortho-quinone yielded several side-chain breakdown products and a trimeric product, in addition to
the cyclization products that eventually yield a melanic pigment [24,25].
It has been reported that DOMA (
electrochemically [28], is converted to DHBAld (
quinone methide intermediate (Figure 4). The reaction proceeds extremely quickly with a half-life of
0.4 s [28]. DOPAC ( )-quinone also rapidly rearranges to give DHBAlc ( ) through a quinone methide
intermediate, and the product DHBAlc ( ) is then gradually converted to DHBAld ( ) through the
same quinone methide as from DOMA ( )-quinone (Figure 4) [29 30]. The present study has confirmed
the results of those previous studies and has characterized the half-lives of DHBAlc ( )-quinone and
DHBAld (7)-quinone, which indicates a high reactivity of the latter with a half-life of 3 min.
DOPAC ( )-quinone and DOMA ( )-quinone do not undergo a cyclization reaction giving a
4)-quinone, generated either enzymatically [26
,27] or
7
) through a decarboxylative rearrangement giving a
3
6
6
7
4
,
6
3
4
five-membered lactone ring in this study. This is in sharp contrast to the behavior of the ortho-quinone
of dihydrocaffeic acid (3,4-dihydroxyphenylpropionic acid), which cyclizes to give a six-membered
lactone ring with a half-life of 9.9 min [31
DOPAC ( )-quinone enzymatically generated and identified 2,5,6-trihydroxybenzofuran, a cyclization
product, in addition to DOMA ( ) and DHBAld ( ). In our study DHBAlc ( ) was nearly quantitatively
,32]. Sugumaran et al. [33,34] previously studied the fate of
3
4
7
6
produced at 2 min (Figure 3E,F). The reason for this discrepancy between their results and ours is not
clear at present. However, one major difference in the reaction conditions is that we used a very high
ratio of tyrosinase to substrate to generate DOPAC (3)-quinone within 1–2 min while they used a much
lower ratio to generate it (>40 min).
The comparison of half-lives between pH 6.8 and 5.3 indicates that the quinone methide
production from DOPE ( )-quinone, DOPEG ( )-quinone and DHBAlc ( )-quinone proceeds through
1
2
6
the base-catalyzed mechanism as previously shown for 4-propyl-o-quinone by Bolton et al. [35].
It appears that the formation of quinone methides is a rather common pathway of ortho-quinone
metabolism, even in the absence of quinone methide tautomerase [27,36]. For example, tyrosinase
oxidation of 4-ethylphenol gives 1-(3,4-dihydroxyphenyl)ethanol as a major product [37]. These
ortho-quinones may exert strong toxicity to neurons through binding with thiol enzymes and the
production of reactive oxygen species in vivo [17–19]. In this regard, it should be worthwhile to point

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out that DHBAld (7)-quinone appears highly reactive as indicated by its short half-life of 3.0 min, the
shortest among the ortho-quinones examined in this study (Table 1).
In summary, the fate of ortho-quinones derived from the catecholamine metabolites DOPE (1),
DOPEG (2), DOPAC (3) and DOMA (4) provided evidence for the facile production of quinone
methides as transient intermediates, which yielded stable products upon aromatization.
4. Experimental Section
4.1. Materials
Tyrosinase (from mushrooms, 1715 U/mg), 6-hydroxydopamine.HCl, DOPEG, DOPAC and
DOMA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The 3,4-dihydroxybenzaldehyde
(DHBAld), 3,4-dihydroxybenzoic acid, ascorbic acid, NaBH4, NaIO4, formic acid, and methanol were
purchased from Wako Pure Chemical Industry (Osaka, Japan). DOPE was purchased from Tokyo
Chemical Industry (Tokyo, Japan). A solution of DHBAlc was prepared by reducing DHBAld with
NaBH4 as described in 4.3. The 2-oxo-2-(3,4-dihydroxyphenyl)ethanol (2-oxo-DOPE) was a kind gift
from Manickam Sugumaran (University of Massachusetts Boston, Boston, MA, USA).
4.2. Instruments
UV-visible spectra were measured with a JASCO V-630 UV-VIS spectrophotometer connected
with an HMC-711 water thermostatted cell holder (JASCO Co., Tokyo, Japan). The HPLC system used
to follow the course of tyrosinase oxidation consisted of a JASCO PU-2080 Plus pump (JASCO Co.,
Tokyo, Japan), a Shiseido C18 column (Capcell Pak MG; 4.6
ˆ
250 mm; 5 µm particle size, Shiseido,
Tokyo, Japan) and a JASCO UV-visible detector at 280 nm (JASCO Co., Tokyo, Japan). The mobile
phases were 0.4 M fo˝rmic acid: methanol, 90:10, 85:15 or 80:20 (v/v) (see Table 1). Analyses were
performed at 35 or 50 C (see Table 1) and a flow rate of 0.7 mL/min. For preparative HPLC to isolate
2-oxo-DOPE, a Shiseido C18 column (Capcell Pak MG; 20
ˆ
250 mm; 5 µm particle size) was used with
˝
a mobile phase of 0.4 M formic acid:methanol (80:20 (v/v)) at 35 C at a flow rate of 7.0 mL/min. The
1H NMR (400 MHz) spectrum was obtained with a Bruker AVANCE 400 spectrophotometer (Billerica,
MA, USA) which was calibrated by adding methanol (3.34 ppm). Mass spectra were obtained using an
Esquire HCT Plus Ion Trap Mass Spectrometer (mode: ESI) (Bruker, Billerica, MA, USA).
4.3. Oxidation of Catecholamine Metabolites with Mushroom Tyrosinase and HPLC Following Oxidation
Stock solutions of metabolites were prepared in ethanol or water at a 10 mM concentration. Test
solutions of metabolites were prepared by diluting them to 100
µM with 50 mM sodium phosphate
buffer (pH 6.8 or 5.3) when needed. A soluti˝on (2 mL) of 100
µM metabolite was oxidized by adding
20 µL (100 U) mushroom tyrosinase at 37 C. The same amount of tyrosinase was also added to
the reference cuvette. The oxidation was followed periodically up to 60 min with the UV-visible
spectrophotometer. For HPLC following oxidation, a solution˝(1 mL) of 100
µM metabolite was
oxidized by adding 20 µL (100 U) mushroom tyrosinase at 37 C. The amount of tyrosinase was
doubled in the HPLC analyses compared to the spectrophotometric analyses, so that oxidation of
ortho-quinones completes faster, because we observed that oxidation of DOMA took several minutes
to complete in the spectrophotometric analysis (Figure 2D). Aliquots of the reaction mixtures were
periodically (2, 5, 10, 15, 30 and 60 min) withdrawn and subjected to analysis after reduction. For
reductive termination of the reaction, 100 µL aliquots were mixed with 20 µL 10% NaBH4 or 5 mM
ascorbic acid in water followed by the addition of 100 µL 0.8 M perchloric acid.
The half-lives (t1/2) of the ortho-quinones produced were calculated using the equation
ln(c0/c) = kt and kt1/2 = 0.693.

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4.4. Preparation of 2-Oxo-DOPE (5)
A solution of DOPEG (2) (17.0 mg, 0.1 mmol) in 100 mL 50 mM sodium phosphate buffer (pH 6.8)
was gently shaken at 37 ˝C, to which NaIO4 (21.4 mg, 0.1 mmol) in 1 mL water was slowly added
in 2 min. After 30 min, the mixture was poured into a solution of ascorbic acid (352 mg, 2 mmol)
in 10 mL water. The mixture was extracted three times with ethyl acetate (100 mL each) and the
combined ethyl acetate extract was dried over anhydrous sodium sulfate. The extract was evaporated
under reduced pressure to give an oily residue, which was subjected to preparative HPLC to afford,
after lyophilization, ca. 0.2 mg (HPLC purity, 98%; NMR purity, 91%) of 2-oxo-DOPE (
5). Tyrosinase
oxidation gave a lower yield of 4.78 (2H, s, CH2), 6.81 (1H, d,
5.
1H NMR (400 MHz, CD3OD)
δ
J = 8.8 Hz, H-2’), 7.36 (1H, dd, J = 8.8, 2.0 Hz, H-6’), 7.37 (1H, d, J = 2.0 Hz, H-5’) (Figure S5). ESI(+)/MS:
m/z 191.03 ([M + Na])+, 33%) 169.05 ([M + H]+, 100%) (Figure S6).
5. Conclusions
This study confirmed the fact that ortho-quinones from catechols 1–4 are converted to quinone
methide tautomers as common intermediates, through proton rearrangement or decarboxylation. The
unstable quinone methides afford stable alcoholic or carbonyl products (Figure 4). In substantia nigra
and locus coeruleus neurons, the production of neuromelanin appears to detoxify otherwise toxic
DA-quinone and NE-quinone [12,14]. Since it is likely that the formation of those ortho-quinones does
not lead to incorporation into neuromelanin, the ortho-quinone of catecholamine metabolites, once
formed, may exert cytotoxicity to neurons.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/17/
2/164/s1.
Acknowledgments: This work was supported by grants from the Japan Society for the Promotion of Science
(JSPS) (No. 24500450, 15K09794) given to Kazumasa Wakamatsu and Shosuke Ito. The authors are very grateful to
Manickam Sugumaran (University of Massachusetts Boston) for his kind gift of 2-oxo-DOPE.
Author Contributions: Conception and design: Shosuke Ito and Kazumasa Wakamatsu; Experiment and
data collection: Yuta Yamanaka, Shosuke Ito and Makoto Ojika; Data analysis and drafting of manuscript:
Yuta Yamanaka, Shosuke Ito and Kazumasa Wakamatsu.
Conflicts of Interest: The authors declare no conflict of interest.
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