Catalytic oxidative cyclocondensation of o-aminophenols to 2-amino-3H-phenoxazin-3-ones

Article abstract of DOI:10.1080/00397910701316136

The catalytic oxidative cyclocondensation of the o-aminophenols 1a-f was investigated. The oxidants used were air/laccase, H2O2/horseradish peroxidase, H2O2/ebselen (3), and TBHP/diphenyl diselenide 4. The products obtained were 2-amino-3H-phenoxazin-3-on

Full text of DOI:10.1080/00397910701316136

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Catalytic Oxidative Cyclocondensation
of o‐Aminophenols to
2
‐Amino‐3H‐phenoxazin‐3‐ones
a
a
a
Mirosław Giurg , Katarzyna Piekielska , Magdalena Gębala , Bartosz
a
a
a
Ditkowski , Marcin Wolański , Wanda Peczyńska‐Czoch & Jacek
a
Młochowski
a
Department of Organic Chemistry, Faculty of Chemistry , Wrocław
University of Technology , Wrocław, Poland
Published online: 30 Jun 2007.
To cite this article: Mirosław Giurg , Katarzyna Piekielska , Magdalena Gębala , Bartosz Ditkowski , Marcin
Wolański , Wanda Peczyńska‐Czoch & Jacek Młochowski (2007) Catalytic Oxidative Cyclocondensation of
o‐Aminophenols to 2‐Amino‐3H‐phenoxazin‐3‐ones, Synthetic Communications: An International Journal for
Rapid Communication of Synthetic Organic Chemistry, 37:11, 1779-1789, DOI: 10.1080/00397910701316136
To link to this article: http://dx.doi.org/10.1080/00397910701316136
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Synthetic Communications , 37: 1779–1789, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701316136
Catalytic Oxidative Cyclocondensation of
o-Aminophenols to
2
-Amino-3H-phenoxazin-3-ones
Mirosław Giurg, Katarzyna Piekielska, Magdalena G e˛ bala,
Bartosz Ditkowski, Marcin Wola n´ ski,
Wanda Peczy n´ ska-Czoch, and Jacek Młochowski
Department of Organic Chemistry, Faculty of Chemistry,
Wrocław University of Technology, Wrocław, Poland
Abstract: The catalytic oxidative cyclocondensation of the o-aminophenols 1a–f was
2 2
investigated. The oxidants used were air/laccase, H O /horseradish peroxidase,
H O /ebselen (3), and TBHP/diphenyl diselenide 4. The products obtained were
2
2
2-amino-3H-phenoxazin-3-one—questiomycin A, its derivative 2b, and cinnabarinic
acid and actinocin (2c,d). Substrates with methyl groups at 4 and 5 positions of
benzene ring were converted to different dihydrophenoxazinones 2g,h. Compounds
having chlorine atoms at the same positions underwent oxidation to planar phenoxa-
zinones 2e,f with elimination of one hydrochloride molecule.
Keywords: o-aminophenols, 2-aminophenoxazin-3-ones, enzymes, hydroperoxides,
organoselenium compounds, oxidation
2
-Amino-3H-phenoxazin-3-one is a part of the chemical structure of actino-
[
1]
mycin D, a known anticancer drug. Compounds having such a skeleton
occur in nature as insect pigments, fungal metabolites, antibiotics, and allelo-
[2,3]
chemicals such as ommochromes, cinnabarines, and questiomycins.
Simple 2-aminophenoxazin-3-ones and 3-aminophenoxazin-2-one exhibit
[3–6]
antitumor, antimicrobial, and antiviral activity in vitro and in vivo.
Received in Poland October 5, 2006
Address correspondence to Mirosław Giurg, Department of Organic Chemistry,
Faculty of Chemistry, Wrocław University of Technology, Wyb. Wyspia n´ skiego 27,
50-370 Wrocław, Poland. E-mail: miroslaw.giurg@pwr.wroc.pl
1
779

1
780
M. Giurg et al.
Aminophenoxazinones have been reported as Streptomyces parvulus
and woodrotting fungi metabolites, cyclocondensation products of o-amino-
phenols with bovine erythrocyte hemolyzate, and bioconversion products of
[
3,6–9]
Pseudomonas putida grown on nitroarenes.
o-aminophenol cyclocondensation by chemical means employed quinones
The traditional method for
or lead and mercury heavy-metal reagents, which are not environmentally
5,10]
[
friendly.
Using hydrogen peroxide activated by cyclodextrin ketone
or dioxygen as the oxidant in the presence of 1-oxyl-2,2,6,6-tetramethylpi-
peridine (TEMPO), cobalt, or copper catalysts, led to 2-aminophenoxazin-3-
[11–13]
one in preparative yield up to 94%.
In our previous article, we reported a convenient method for oxidative
cyclocondensation of o-aminophenol to 2-amino-3H-phenoxazin-3-one
(
questiomycin A) with air-activated by Co(salen), t-butylhydroperoxide
0
(
hydrogen peroxide catalyzed by ebselen.
TBHP) catalyzed by 3,3 -ditrifluoromethyldiphenyl diselenide, or
It corresponds to the modern
[14]
trends in organic synthesis where the dioxygen, hydrogen peroxide, and
TBHP are used as the reagents because they are cheap and environmen-
[15]
tally friendly and are used on both laboratory and industrial scales.
Nevertheless, because these reagents are generally inactive or unselective
toward most of the organic substrates, suitable activators must be
[
12–16]
used.
In this work, we concentrated our study on the oxidative coupling of
different ring-substituted o-aminophenols 1a–f to aminophenoxazinones
2a–f in both enzymatic and nonenzymatic conditions. The laccase was a
catalyst for aerobic oxidation, whereas horseradish peroxidase was a
catalyst for hydrogen peroxide oxidation. In the nonenzymatic conditions,
the hydrogen peroxide activated by ebselen (2-phenylbenzisoselenazol-
0
3
(2H)-one) (3) and t-butylhydroperoxide in the presence of 3,3 -ditrifluoro-
methyldiphenyl diselenide (4) were used as reagents. Although the results
strongly depended on the structure of substrate, oxidant used, and reaction
conditions, the reaction has a synthetic value because of appreciable selectiv-
ity and moderate to high yields of the products.
The aminophenols taken as the substrates fall into two different groups.
There were o-aminophenol (1a) and its derivatives, having substituents in
the vicinity to the amino and hydroxy groups 1b–d, and o-aminophenols
substituted at remote ring carbon atoms 1e–h. The compounds 1a–h
were oxidized by chemical means with H O /3 (method A) or TBHP/4
2
2
(
method B), or enzymatically with air/laccase (method C) or H O /horse-
2 2
radish peroxidase (method D). The results are presented in Scheme 1 and
Table 1.
In all cases, the conversion of the substrates was complete (except
carboxy derivatives 1c), and desired products were isolated in the yields
given in Table 1. The results of cyclocondensation depended on the
substrate structure. The yields of the each product differed depending on the
oxidant used.

2-Amino-3H-phenoxazin-3-ones
1781
Scheme 1.
In the chemical method, the reaction was carried out in t-butanol for 20 h,
and the catalyst was used in the 5% molar amount (3,4). The reaction con-
ditions were optimized. The reaction was faster when the molar ratio of
H O to the substrate 1a was 4:1 and TBHP to 1a was 5:1. In this way, o-amino-
2
2
phenol (1a) gave questiomycin A (2a) in good to excellent yield. 2-Amino-3-
methylphenol (1b) was smoothly oxidized to the 1,9-dimethyl derivative of
questiomycin A 2b because the methyl group at the vicinity of the amino
group strongly activates the substrate molecule. The product 2b was formed
in an excellent 93% yield even when the catalyst 3 was used in the amount
of 0.10 mol %. In contrast, a strong electron-withdrawing carboxy substituent
present at the same position of aminophenol 1c inhibited the reaction, and cin-
nabarinic acid (2c) was not formed. The electron-donating methyl group in the

1
782
M. Giurg et al.
Table 1. Results of the oxidative cyclocondensation of o-aminophenols 1a–h
Reaction
time (h)
a
Substrate
Method
Product
Yield (%)
92
1a
1b
1c
A
B
20
20
2a
b
83
29 (39)
b
C
4.5
20 min
20
b
D
(40)
A
2b
2c
92
64
B
20
23
C
24 (43)
(52)
—
D
40 min
20
22
A or B
C
38 (42)
38
81
D
15
20
1
d
e
A
2d
2e
C
1
20
(53)
44
1
A
B
20
42
C
3.5
20
51 (54)
—
27
1
1
f
A
2f
B
20
C
7
20
35 (36)
c
42
g
A
2g
B
20
55
72 (84)
(61)
C
0.5
0.5
20
D
1h
A
2h
1.5
15
B
20
C
1
0.5
60 (67)
(15)
D
a
Preparative yield. Data in parantheses refer to yields determined by UV/VIS
analysis.
b
[14]
See lit.
c
3
eq. of hydrogen peroxide was used (30%, 0.60 ml, 6.0 mmol).
vicinity to the hydroxyl group of 2-amino-3-carboxy-6-methylphenol 1d made
the substrate susceptible to cyclocondensation, and actinocin (2d) was the sole
product. The chlorine atoms at remote ring carbon atoms in compounds 1e,f
entailed cyclocondensation to 2-aminophenoxazinones 2e,f with elimination
of one hydrochloride molecule. Methyl substituents present at the same
positions in the aminophenols 1g,h made the products more complex. The
different nonplanar dihydrophenoxazinones 2g and 2h were produced in satis-
factory to good yield. Generally, the yield of the produced phenoxazinones
strongly depended on the electron character and position of substituents
used. The results are given in Table 1.

2-Amino-3H-phenoxazin-3-ones
1783
Cyclocondensation of o-aminophenol and its derivatives in the presence
of biocatalysts carried out in a phosphate buffer at room temperature
yielded similar products. Enzymatic oxidation of o-aminophenol (1a) and
its derivatives substituted in the vicinity of the amino group with electron-
donating methyl group 1b and electron-withdrawing carboxy group 1c,d
gave appropriate 2-aminophenoxazinones with quite satisfactory yield.
o-Aminophenols with methyl groups substituted at remote ring carbon atom
1
g,h were converted to dihydrophenoxazinones 2g and 2h even up to 84%,
and analogously substituted chloroaminophenols 1e,f in aerobic condition
method C) gave 7- or 8-chloro-2-aminophenoxazinones 2e and 2f. A
(
similar oxidation pattern of aminophenols by chemical means was observed.
The results presented here support our earlier hypothesis that formation of
aminophenoxy radicals by oxidation of o-aminophenols by hydroperoxides in
the presence of organoselenium compounds are crucial steps in their conver-
[
14]
sion to 2-aminophenoxazin-3-ones.
with well-known mechanism of action of laccase and peroxidase and other
Our findings are also in accordance
[
17]
metaloenzymes.
The cyclocondensation of o-aminophenols can be used successfully for
preparation of phenoxazinones and dihydrophenoxazinones. The yield of the
products strongly depends on the substrates and oxidant used. For
the enzymatic method, more suitable is the air/laccase system, and for nonenzy-
matic conditions, more efficient as oxidant is hydrogen peroxide activated by
[18]
ebselen. Both recommended catalysts are easy to obtain in the laboratory.
We expect that 2-aminophenoxazinones with a planar structure of three
condensed rings will be able to intercalate into cellular DNA similar to that
[1]
described for anticancer actinomycin D drug. Althought planar phenoxazinones
and nonplanar dihydrophenoxazinones are suspected to be biologically active
similar to that reported in Refs. 4–6, 8, and 19, the biological screening of
compounds 2 and derivatives as potential cytostatics and virucides is in progress.
EXPERIMENTAL
Melting points were determined on a digital melting-point apparatus, Electro-
1
13
thermal IA 91100. H NMR and C NMR were measured with a Bruker DRX
00 spectrometer. IR (KBr pallets) was measured on a Perkin-Elmer 2000 FT
3
spectrometer. UV/VIS was measured on a Jasco V-530 spectrofotometer.
Because the physicochemical data reported in the previous papers had been
incorrect, they are carefully revised.
t-Butylhydroperoxide (80% in di-tert-butylperoxide/water 3:2), 30%
hydrogen peroxide, o-aminophenols 1a–c and 1e–h, horseradish peroxidase,
and other commercial reagents and solvents were purchased from Aldrich,
Fluka, or Sigma Co. Water to enzymatic transformations was distilled twice
before using. Laccase was prepared as reported earlier in Ref. [18a].
2
-Amino-3-hydroxy-4-methylbenzoic acid (1d) was prepared by reduction of

1
784
M. Giurg et al.
nitro group of 3-hydroxy-4-methyl-2-nitrobenzoic acid with KBH in the
4
presence of Pd/C. 2-Phenylbenzisoselenazol-3(2H)-one (ebselen) was
[
18b]
0
prepared from anthranilic acid.
3) was delivered by Ludwik Syper from our laboratory. The purity of the
products was confirmed by comparison of their melting point with data given
3,3 -Ditrifluoromethyldiphenyl diselenide
(
[
2a]
[20]
[12]
in literature for 2a, 2b,
UV/VIS spectra.
and 2e,g,
and measuring their NMR, IR, and
Preparation of 2-Amino-3-hydroxy-4-methylbenzoic Acid (1d)
To a suspension of 5% Pd/charcoal (50 mg) in distilled water (5.0 ml),
methanol (10 ml), 2 drops of 4% sodium hydroxide in water and KBH4
(
500 mg, 9.27 mmol), 3-hydroxy-4-methyl-2-nitrobenzoic acid (0.986 g,
.0 mmol) was added portionwise at 35–408C over 20 min. The reaction
was continued for an additional 2 h, filtered, and washed with methanol
5 ml). The filtrate was acidified with 2 M HCl to pH ca. 1 and washed
5
(
with diethyl ether (3 ꢀ 150 ml). pH was adjusted to ca. 2.0 with sodium
hydroxide and extracted with diethyl ether. The combined extracts were
dried over anhydrous sodium sulfate; solvent was distilled off to give
pure 2-aminophenol 1d (0.710 g, 4.25 mmol, 85%). Mp 234–2368C
(
cm
hydrochloride of 1d mp 165–1678C, ref. [20] 162–1638C). v
(KBr)
3400–2000 (broad, COOH, NH , OH), 1653 or 1612 (C 55 O),
max
2
1
2
1
1274, 1234, 1210 (C-N and/or C-O); H NMR (DMSO-d ) 10.11
6
3
(
s, 1H, COOH), 8.29 (s, 2H, NH ), 7.20 (d, J ¼ 8.1 Hz, 1H, H-6), 6.31
2
3
(
d, J ¼ 8.1 Hz, 1H, H-5), 2.31 (s, 1H, OH), 2.15 (s, 3H, Me).
Oxidation of o-Aminophenols 1a–h by Chemical Means
To a mixture of o-aminophenol (1a–h) (2.0 mmol) and a selenium catalyst,
ebselen (3) (for H O ) or 4 (for TBHP) (0.10 mmol) in t-butanol (10 ml),
2
2
30% H O (0.8 ml, 8.0 mmol), or TBHP (1.25 ml, 10 mmol) was added.
The reaction mixture was magnetically stirred at room temperature (H O )
2 2
2
2
or at 558C (TBHP) for 20 h. After this period, the reaction was stopped by
addition of a pinch of Pt/C and the solution of NaHCO (1.25 g) and NaCl
(
3
4.0 g) in water (50 ml), and the mixture was vigorously stirred at room temp-
erature until evolution of carbon dioxide and dioxygen ceased. The product
was extracted with chloroform (until the red color disappear) and dried over
anhydrous Na SO . Silica gel (70–230 mesh, 7.5 g) was added to the
2
4
solution. The solvent was removed in vacuo, the residue was poured into a
silica-gel column (70–230 mesh, 20 g), and products 2a,b,e,f,h were
isolated by eluting with chloroform–ethyl acetate (5:1) and 2g with
gradient (5:1–2:1). Actinocin (2d) crystallized directly on the reaction
mixture and was filtered, washed with t-butanol, and dried in the air. The
results are given in Table 1.

2-Amino-3H-phenoxazin-3-ones
1785
Data
2
-Amino-3H-phenoxazin-3-one (2a) (Questiomycin A): Red powder. Mp
1
2
588C (ref. [2a,12] mp 256–2588C). H NMR (DMSO-d ): 7.70 (dd,
6
3
4
3
4
J ¼ 7.8 Hz, J ¼ 1.5 Hz, 1H, H-9); 7.50 (dd, J ¼ 8.2 Hz, J ¼ 1.9 Hz, 1H,
3
3
4
H-6); 7.45 (ddd, J ¼ 8.2 Hz, J ¼ 6.8 Hz, J ¼ 1.5 Hz, 1H, H-7); 7.38
3
3
4
(
ddd, J ¼ 7.8 Hz, J ¼ 6.8 Hz, J ¼ 1.9 Hz, 1H, H-8); 6.83 (s, 2H, NH );
2
6
.36 (s, 1H, H-1 or H-4); 6.34 (s, 1H, H-1 or H-4). IR (KBr): g ¼ 3413,
2
2
306 cm (NH ), 1587 cm (br.) (C 55 O, C 55 N), 1273, 1203, 1115 cm
1
21
21
3
(C-N, C-O). UV/VIS (methanol): lmax ¼ 434 nm (log 1 ¼ 4.252).
2
-Amino-1,9-dimethyl-3H-phenoxazin-3-one (2b): Red powder. Mp 2478C
1
(ref. [21] mp 2338C). H NMR (DMSO-d ): 7.20–7.40 (m, 3H, H-6, H-7,
6
H-8); 6.35 (s, 2H, NH ); 6.23 (s, 1H, H-4); 2.59 (s, 1H, 1-CH or 9-CH ); 2.22
2
3
3
2
1
(
s, 3H, 1-CH or 9-CH ). IR (KBr): v ¼ 3462, 3389, 3359, 3248 cm (NH2
3
3
21
21
and N-H of tautomer), 1550–1639 cm (C 55 O, C 55 N), 1250 cm (C-N),
21
1
201 cm (C-O). UV/VIS (methanol): lmax ¼ 422 nm (log 1 ¼ 4.379).
2
-Amino-1,9-dicarboxy-4,6-dimethyl-3H-phenoxazin-3-one (2d) (Actinocin):
1
Dark red powder. Mp 259.5–2608C (ref. [18a] mp not reported). H NMR
3
(
DMSO-d ): 9.68 (s, 2H, COOH), 8.76 (s, 2H, NH ), 7.85 (d, J ¼ 8.0 Hz, 1H,
6
2
3
H-8), 7.47 (d, J ¼ 8.0 Hz, 1H, H-7), 2.50 (s, 3H, 4-CH or 6-CH ), 2.12
3 3
21
(
s, 3H, 4-CH or 6-CH ). IR (KBr): v ¼ 2000–3400 cm (COOH and OH),
3
3
21
21
390, 3251 cm (NH ), 1678 cm (C 55 O), 1583 cm (C 55 N), 1324, 1293,
225 cm
¼ 4.439).
21
3
1
1
2
21
(C-N and/or C-O). UV/VIS (methanol): lmax ¼ 430 nm (log
2
-Amino-7-chloro-3H-phenoxazin-3-one (2e): Red powder. Mp 296–2978C
1 3
(
ref. [12] mp 2888C). H NMR (CDCl ): 7.70 (d, J ¼ 8.6 Hz, 1H, H-9), 7.65
3
4
3
4
(d, J ¼ 2.2 Hz, 1H, H-6), 7.43 (dd, J ¼ 8.6 Hz, J ¼ 2.2 Hz, 1H, H-8), 6.87
2
1
(s, 2H, NH ), 6.36 (s, 2H, H-1 and H-4). IR (KBr): v ¼ 3453 and 3364 cm
2
21
21
(NH ), 1610 (broad) and 1578 cm (C 55 O and C 55 N), 1284, 1181, 1073 cm
2
(C-N and/or C-O). UV/VIS (methanol): lmax ¼ 436 nm (log 1 ¼ 4.424).
2
-Amino-8-chloro-3H-phenoxazin-3-one (2f): Red powder. Mp 270–2718C
1
4
(
CHCl ). H NMR (DMSO-d ): 7.74 (d, J ¼ 2.4 Hz, H-9); 7.54
3
6
3
3
4
(
d, J ¼ 8.8 Hz, 1H, H-6); 7.47 (dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz, 1H, H-7);
7
.01 (s, 2H, NH ); 6.38 (s, 1H, H-1 or H-4); 6.32 (s, 1H, H-1 or H-4). IR
2
2
1
21
(
KBr): v ¼ 3345, 3367 cm (NH ), 1607(br.), 1572 cm (C 55 O, C 55 N),
2
2
1
1
207, 1188, 1072 cm
(C-N, C-O). UV/VIS (ethanol): lmax ¼ 439 nm
12 7 2 2
(
log 1 ¼ 4.345). Found: C, 58.04; H, 3.10; Cl, 14.55. (C H N O Cl)
2
46.65 requires C, 58.44; H, 2.86; Cl, 14.37.
2
2
-Amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazin-3-one (2g): Mp
1
318C (ref. [12] mp 178.5–179.58C). H NMR (DMSO-d ): 7.08
6

1
786
M. Giurg et al.
3
3
4
(
d, J ¼ 7.9 Hz, 1H, H-9); 6.79 (dd, J ¼ 7.9 Hz, J ¼ 1.0 Hz, 1H, H-8); 6.71
4
(
d, J ¼ 1.0 Hz, 1H, H-6); 6.36 (s, 2H, NH ); 6.04 (s, 1H, H-1), 3.19
2
2
2
(
d, J ¼ 15.9 Hz, 1H, H-4a); 2.97 (d, J ¼ 15.9 Hz, 1H, H-4b); 2.25 (s, 3H,
2
1
7
-CH ); 1.09 (s, 3H, 4a-CH ). IR (KBr): v ¼ 3401, 3288, 3165 cm (NH
3
3
2
2
1
and OH of tautomer), 1695, 1623 cm
(
(C 55 O, C 55 N). UV/VIS
methanol): lmax ¼ 400 nm (log 1 ¼ 4.144).
3
(
-Amino-1,4a-dihydro-4a,8-dimethyl-2H-phenoxazin-2-one (2h): Mp 2048C
1
ref. [5] not reported). H NMR (CDCl ): 10.03 (s, 2H, NH ); 7.05 (s, 1H, H-9);
3
2
2
6
.88 (s, 2H, H-6, H-7); 6.14 (s, 1H, H-4); 3.33 (d, J ¼ 15.2 Hz, 1H, H-1a); 3.26
2
(d, J ¼ 15.2 Hz, 1H, H-1b); 2.34 (s, 3H, 8-CH ); 1.42 (s, 3H, 4a-CH ). UV/VIS
3
3
(methanol): l ¼ 349 nm (log 1 ¼ 4.250).
max
Laccase-Catalyzed Aerobic Oxidation of o-Aminophenols (1a–h)
Aminophenol 1a–h (0.50 mmol) in methanol (2 ml) was added to a vigorously
stirred solution of laccase (0.6U for 1b,e,f; 3U for 1h; 4U for 1a; 5U for 1g; 6U
[
22]
for 1c; 10U for 1d)
in 5.0 pH phosphate buffer (100 ml, 0.066 M,
Na HPO /KH PO ) in an open vessel to supply oxygen for the period given
2
4
2
4
in Table 1. The reaction was monitored using thin-layer chromatography
TLC). After the reaction was finished, products 2a,b and 2e–h were
extracted with ethyl acetate, dried other anhydrous Na SO , and purified on
(
2
4
a silica-gel column as described by chemical means (in this article).
For isolation of cinnabainic acid (2c) and actinocin (2d), the reaction
mixture was acidified to pH ca. 2 with 0.15 M hydrochloric acid. The
formed precipitate was washed several times with distilled water,
collected by centrifugation, and crystallized from methanol–water to yield
pure acids 2c,d. The results are given in Table 1. Spectroscopic data are
recorded.
Data
2
-Amino-1,9-dicarboxy-3H-phenoxazin-3-one (2c) (cinnabarinic acid):
1
Dark red powder. Mp . 3308C (ref. [18a] mp not reported). H NMR
3
(
DMSO-d ): 9.71 (s, 1H, COOH); 8.79 (s, 1H, COOH); 7.95 (dd, J ¼ 7.1 Hz,
6
4
3
4
J ¼ 1.1 Hz, 1H, H-6 or H-8); 7.77 (dd, J ¼ 7.7 Hz, J ¼ 1.1 Hz, 1H, H-6 or
3
3
H-8); 7.60 (dd, J ¼ 7.7 Hz, J ¼ 7.1 Hz, 1H, H-7); 6.60 (s, 1H, H-4); 5.75
(
s, 2H, NH ). UV/VIS (ethanol): l
¼ 450 nm (log 1 ¼ 4.305).
2
max
Horseradish Peroxidase–Catalyzed Hydrogen Peroxide Oxidation
of o-Aminophenols
[23]
To the magnetically stirred solution of horseradish peroxidase (4U)
in pH
5
.0 phosphate buffer (70 ml, 0.066 M, Na PO /KH PO ), o-aminophenol (1)
2
4
2
4

2-Amino-3H-phenoxazin-3-ones
1787
(
0.18 mmol) in methanol (1.0 ml) and 30% H O (0.090 ml, 0.90 mmol) were
2
2
added. The reaction was monitored by TLC. When the substrate vanished, the
products were extracted with ethyl acetate and dried over anhydrous Na SO .
2
4
Solvent was removed, and the residue was dissolved in ethanol. The amounts
of 2a,b,g,h were estimated by UV/VIS spectroscopy as the data given in
chemical means and aerobic oxidation (in this article). For 2c, the reaction
mixture was acidified to pH 2 with hydrochloric acid, and the product was
extracted with ethyl acetate to yield dark red powder of cinabarinic acid.
The results are given in Table 1.
ACKNOWLEDGMENTS
0
The authors thank Ludwik Syper for the 3,3 -ditrifluoromethyldiphenyl dise-
lenide catalyst (4) used in this work. This work was supported by the Polish
State Committee for Scientific Research (Grant No. 3T09A 003 27).
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