Regioselective synthesis of 3-hydroxyorthanilic acid and its biotransformation into a novel phenoxazinone dye by use of laccase

Article abstract of DOI:10.1002/ejoc.200700865

A natural phenoxazinone derivative, cinnabarinic acid (2, CA, R = CO ₂H), could be obtained in vitro with the aid of purified laccase from the fungi Pycnoporus cinnabarinus by oxidative dimerization of 3-hydroxyanthranilic acid (1, 3-HAA, R = C

Full text of DOI:10.1002/ejoc.200700865

FULL PAPER
DOI: 10.1002/ejoc.200700865
Regioselective Synthesis of 3-Hydroxyorthanilic Acid and Its Biotransformation
into a Novel Phenoxazinone Dye by Use of Laccase
Frédéric Bruyneel,[a] Estelle Enaud,[b] Ludovic Billottet,[a] Sophie Vanhulle,[b] and
Jacqueline Marchand-Brynaert*[a]
Keywords: Laccase / Phenoxazinone / Lithiation / Biotransformation / Aromatic sulfonation reaction
A natural phenoxazinone derivative, cinnabarinic acid (2,
CA, R = CO2H), could be obtained in vitro with the aid of
purified laccase from the fungi Pycnoporus cinnabarinus by
oxidative dimerization of 3-hydroxyanthranilic acid (1, 3-
HAA, R = CO2H). With the aim of gaining access to a new
class of water-soluble chromophores and potential bioactive
molecules, the regioselective sulfonation of 2-hydroxyaniline
was investigated. Straightforward insertion of a sulfonate
group into a carbon–metal bond provided an efficient and
selective process for the synthesis of 3-hydroxyorthanilic acid
(3, 3-HOA, R = SO3H). This sulfonated compound was then
subjected to laccase oxidation in order to mimic the cinna-
barinic acid synthesis. A practical HPLC method to study the
oxidized products was developed, and the major product of
biotransformation – namely 2-amino-3-oxo-3H-phenoxazin-
1,9-disulfonic acid (4, LAO, R = SO3H) – was isolated and
identified as the sulfonate analogue of cinnabarinic acid.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
The search for water-soluble long-wavelength fluorochro-
mophores for the detection of biological and organic mole-
cules has recently attracted great interest. Indeed, fluores-
cence-based techniques are sensitive, simple and selective.
In particular, phenoxazinone chromophores represent po-
tentially good leads for the development of fluorescent
probes for the detection of hydroxyl radicals.[5] These radi-
cals are largely responsible for DNA damage by endoge-
nous effects but also by several indirect effects.[6] However,
the poor solubilities of the currently available phenoxazin-
one derivatives represent a drawback for biological appli-
cations. One way to make these molecules soluble in aque-
ous media is to add a highly polar substituent such as a
sulfonate group.
Introduction
Cinnabarinic acid (2) is a red phenoxazinone compound
responsible for the colour of the Pycnoporus species.[1–3]
White rot fungi produce cinnabarinic acid from 3-hydroxy-
anthranilic acid (1, 3-HAA) through oxidative dimerization
catalysed by laccase (Scheme 1). Furthermore, phenoxazin-
ones have also been identified in various other biological
systems, including pigments and antibiotics. Actinomycin,
for instance, is an antibiotic formed by phenoxazinone syn-
thase, an enzyme from Streptomyces species.[1] Other phen-
oxazines of industrial interest include the Meldola Blue and
Nile Red dyes.[4]
To the best of our knowledge, selective sulfonation at a
specific position in phenoxazinones has never been reported
before. From the point of view of direct sulfonation of the
phenoxazinone core, the extent and selectivity of the reac-
tion may be hard to control. Similar reactions for the syn-
thesis of sophisticated analogues of 3H-phenoxazin-3-one
by halogenation failed.[7–9]
Scheme 1. Biomimetic synthesis of phenoxazinone dyes.
Furthermore, the usual chemical synthesis of phenoxaz-
ine derivatives involves the condensation of nitroso com-
pounds at elevated temperatures.[10–12] Such processes are
highly toxic and energy-consuming. Moreover, the cycliza-
tion of sulfonated nitrosoanilines as building blocks is not
very effective.[12] On the other hand, it is possible to intro-
duce a sulfonic group regioselectively onto a phenoxazine
precursor[13] that could undergo further oxidative coupling
in the presence of the appropriate catalyst.[2,3]
[a] Université Catholique de Louvain, Organic and Medicinal
Chemistry Unit (CHOM),
Place Louis Pasteur 1, bte. 2, 1348 Louvain-la-Neuve, Belgium
Fax: +32-10-474168
E-mail: marchand@chim.ucl.ac.be
[b] Université Catholique de Louvain, Microbiology Unit
(MBLA),
Place Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
72
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 72–79

Novel Phenoxazinone Dye
Nowadays, the use of enzymes integrated with traditional before with unsubstituted N-protected aniline,[13] would be
synthetic methods in order to develop more environmen- the most straightforward strategy for producing 3-HOA.
tally friendly processes is a popular field of study.[14,15] The
Our selected synthetic path thus starts from commercial
aim of this particular study was to investigate the biomime-
tic synthesis of a sulfonated phenoxazinone derivative based
on the use of laccase. A regioselective sulfonation method
to synthesize 3-hydroxyorthanilic acid (3-HOA), the sul-
fonic analogue of 3-hydroxyanthranilic acid, was developed,
and the laccase-catalysed oxidation of this compound was
studied further.
o-aminophenol, which already has the required substituents
for a dimerization process catalysed by laccase (Scheme 1).
A minimal number of steps, inexpensive protecting groups
and purification methods are required for further scaling
up. Pivaloyl amide was chosen to protect the amine, and a
few classical protecting groups (alkyl and silyl ethers) were
tested for masking the phenol. Through this preliminary
screening, the two combinations of methyl ether/pival-
amide[24,25] (5a) and triisopropylsilyl ether/pivalamide (5b)
were selected for the development of a direct sulfonation
process (Scheme 2).
Results and Discussion
Synthesis of 3-Hydroxyorthanilic Acid (3-HOA)
Metallation of 5 followed by quenching of the dianion
The sulfonation reaction is a rather old chemical process. intermediate with methyl iodide was first studied in order
Sulfonic acids clearly impact on industrial chemistry in to optimize the experimental conditions and to control the
various subfields of organic synthesis. They are used as pro- regioselectivity (Table 1). Deprotonation of 5a at –78 °C in
tecting groups, as ligand modifiers[16,17] and as bioisosteres THF with the nBuLi/TMEDA couple or with the more ba-
in medicinal chemistry.[18–20]
sic tBuLi failed, with the starting material being recovered
Direct sulfonation of aromatic compounds is still the (entries 1–2). A similar result was obtained with 5b (en-
most common approach used when the aryl core is rather try 3). When a solution of 5a and tBuLi was warmed up
complex. The regiochemistry is then controlled by the ring to –20 °C before addition of MeI at the same temperature,
substituents. Another efficient method for polysubstituted however, methylation products 6a and 7a (Scheme 2) were
aromatic compounds proceeds through the production of a formed in 7:2 ratio and with a conversion rate of 90% (en-
sulfonated intermediate from a commercial sulfonic acid. try 4). At –10 °C, the regioselectivity in favour of the de-
This intermediate is then broken down in a selective way. sired regioisomer 6a was enhanced to 8:1 (entry 6). In the
Using such reactions, 3-HOA (3) has previously been re- case of the more hindered precursor 5b, the yield was
ported as a by-product from the reduction of an azoic slightly lower (entry 5) at –20 °C and the reaction could not
dye.[21]
be performed at –10 °C, due to the instability of the silyl
However, sulfonation regiochemistry can be also con- ether. Raising the temperature of the dianion solution
trolled either by modulating the reactivity of sulfur trioxide seems to allow the equilibration of 3Li-5 and 6Li-5 interme-
by means of dioxane,[22] or by using a silane substituent diates, favouring the dianion stabilized by intramolecular
already present on the ring to direct the insertion reac- complexation. Since nBuLi is better suited than tBuLi to
tion.[23] The ipso substitution of arylsilanes by various elec- handling at room temperature,[26] the next reactions were
trophiles has recently led to numerous interesting transfor- performed with the less basic nBuLi. Deprotonation of 5a
mations, but this method first requires the introduction of at 0 °C to 25 °C and quenching at 25 °C thus furnished an
a silane group at the correct position through an ortho-di- 80% yield of 6a, without detectable regioisomer 7a (en-
rected metallation reaction. Therefore, the direct insertion try 7). Similarly, the yield of 6b (entry 8) was 70%. The fol-
of oxidized sulfur into the carbon–metal bond, as reported lowing conditions were therefore retained for deprotonation
Scheme 2. Direct sulfonation: quenching of the dianion intermediate. Reagents and conditions: a) 1.1 equiv. tBuCOCl, 3 equiv. NaHCO3,
H2O/EtOAc, 25 °C 4 h; b) 1.1 equiv. MeI, 1.1 equiv. K2CO3, DMF, 0 °C, 1 h then 25 °C, 8 h; c) 2.4 equiv. nBuLi, THF, 0 °C, 10 min, then
25 °C, 3 h; d) 1.5 equiv. C3H9N.SO3, –10 °C to 0 °C, 2 h, then 25 °C, 15 h; e) 0.9 equiv. HSO4·N(Bu)4; f) anion-exchange resin IRA-900,
MeOH/H2O, then HCl/H2O; g) 3 equiv. BCl3, CH2Cl2, –20 °C, 1 h, then 25 °C, 8 h; h) 0.5 equiv. TFA, MeOH, 80 °C, 3 h; i) HCl (6 ),
80 °C, 20 h; j) HBr (48% in water), 120 °C, 17 h.
Eur. J. Org. Chem. 2008, 72–79
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73

J. Marchand-Brynaert et al.
FULL PAPER
Table 1. Experimental conditions for ortho-metallation and quenching.[a]
–
E = Me[e]
E = SO3 M+[f]
Entry
(series)
[c]
[d]
Base (2.4 equiv.)
T0, T1
T2
5
6
7
Others[g]
5
8
1 (a)
2 (a)
3 (b)
4 (a)
5 (b)
6 (a)
7 (a)
8 (b)
9 (a)
10 (a)
11 (a)
12 (b)
nBuLi/TMEDA
tBuLi
–78, –78
–78, –78
–78, –78
–78, –20
–78, –20
–78, –10
0, 25
0, 25
0, 25
0, 25
0, 25
–78
–78
–78
–20
–20
–10
25
–20
–78
–20
25
100%
100%
80%
10%
20%
10%
20%
20%
–
–
–
–
–
–
20%
–
10%
–
–
–
–
20%
–
30%
–
–
10%
–
–
–
10%
tBuLi
tBuLi
tBuLi
tBuLi
nBuLi
nBuLi
nBuLi
nBuLi
70%
50%[b]
80%[b]
80%[b]
70%[b]
85%
30%
40%
40%
15%[b]
70%[b]
60%[b]
50%[b]
nBuLi
nBuLi
0, 25
–20
[a] NMR yields in CDCl3: ratios of conversion were determined by comparison of residual proton signals for H-3 (δ = 8.42 and 8.44 ppm
respectively for 5a and 5b) with H-4 (δ = 6.83 ppm) and CH3 (δ = 2.20 ppm) for 6a; with H-3 (δ = 8.22 ppm) and CH3 (δ = 2.30 ppm)
1
for 7; with H-4 (δ = 6.81 ppm) and CH3 (δ = 2.17 ppm) for 6b. [b] Products isolated and structures confirmed by MS, H NMR and 13C
NMR spectroscopy. [c] The base was allowed to react with 5a–b at temperature T0 for 15 min, then for 3 h at temperature T1 (°C). [d]
The dianion was allowed to react with the electrophile for 2 h at temperature T2 (°C). [e] Quenching was achieved by addition of 1 equiv.
of MeI to the dianion solution. [f] Quenching was achieved by addition of the dianion solution to a suspension of sulfur electrophile
(SO3·C3H9N); the reaction time of 2 h was extended by 15 h at 25 °C. [g] Products arising from partial deprotection of the silyl ether;
TMEDA = tetramethylethylenediamine.
of 5: treatment at low temperature (Յ0 °C) with nBuLi
(2.4 equiv.) in THF, then further reaction at 25 °C for 3 h.
Insertion of oxidized sulfur into a carbon–metal bond is
usually achieved with gaseous sulfur dioxide or trioxide.
While such reagents are troublesome to use, solid sulfur tri-
oxide complexes[27] are much easier to handle and in general
give good yields. Synthesis of the sulfonated intermediate 8
(M+ = Li+) was performed by addition of the dianion solu-
tion to SO3·trimethylamine complex as the electrophile,
firstly at low temperature (Table 1). Quenching of 3Li-5a at
–78 °C was unsuccessful (entry 9), while quenching between
–20 °C and +25 °C furnished a 60–70% yield of sulfonate
8a (entries 10–11) but only a 50% yield of sulfonate 8b (en-
try 12), without detectable contamination by the 6-regioiso-
mers.
Scheme 3. Optimised synthesis of 3-hydroxyorthanilic acid.
Hence, precursor 5a was selected for large-scale synthesis
(Scheme 3). The dianion was produced under the optimized
conditions, this solution then was added to SO3·Me3N at exchange resin bearing quaternary ammonium groups was
–10 °C, and the reaction mixture was kept at low tempera- chosen to quench the aromatic sulfonate. Deprotonated 9
ture for two hours before being allowed to run overnight at could be trapped on the column cationic residues and puri-
room temperature. Isolation of the sulfonic acid was fied by removing all other organic compounds. Free sul-
achieved by forming its tetrabutylammonium salt 8a (M+ = fonic acid 9 was then eluted in quantitative yield. Sulfonic
Bu4N+), a method inspired by the production of monobac- acids are often poorly soluble in organic solvents; neverthe-
tam antibiotics.[28] The following steps of Scheme 3 involved less the methyl ether cleavage of 9 with BCl3 could be
the so-called “cosmetic reactions”.
worked up in dichloromethane. Purification by filtration
or BCl3 in through RP18 silica gel yielded the desired compound.
[29]
Methyl ether cleavage in 8a with BBr3
dichloromethane gave the expected product in high yield. Interestingly, it was present as a 7:3 mixture of noncyclized
However, subsequent hydrolysis of the pivalamide, either in (10) and cyclized (11) product. The presence of the sulfonic
strongly acidic or in basic media, was sluggish and the isola- acid in the ortho position seems to activate the amide func-
tion of the final product by chromatography on reversed tion towards intramolecular nucleophilic attack from the
phase rather difficult, due to the presence of unidentified phenol and the loss of a water molecule. Total cyclization
by-products. This prompted us to investigate the use of a into 2-tert-butylbenzoxazole-4-sulfonic acid (11) was
less expensive process to isolate the free sulfonic acid 9 be- achieved by gentle heating in the presence of a catalytic
fore performing the deprotections (Scheme 3). An anion- amount of trifluoroacetic acid. Compared with the chal-
74
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Eur. J. Org. Chem. 2008, 72–79

Novel Phenoxazinone Dye
lenging synthesis of benzoxazole-4-carboxylic deriva-
tives,[19,30] this sulfonic analogue offers a promising means
of access to a new class of valuable intermediates for fine
chemicals.
The last step of our synthesis consisted of a clean acidic
hydrolysis of the imidate bond of 11 with HCl (6 ). Alter-
natively, simultaneous deprotection of both functions could
be achieved from precursor 9 by heating in aqueous HBr[31]
solution. The final target 3-HOA was obtained with an
overall yield of 50%. The purity of compound 3 as deter-
mined by HPLC was about 95%.
The structures of the sulfonated products 8–11 and 3
were unambiguously determined by NMR spectroscopy.
The question of whether the sulfonate group had been in-
serted into the position ortho to the amino or to the hy-
droxy group was best answered by HMQC and HMBC
spectra performed on 3 (3-HOA). The signals of the aro-
matic protons H-4, H-5 and H-6 were assigned to their rela-
tive carbons by HMQC. A J3 correlation between the car-
bon at position C-2 (substituted with NH2) and the two
proton signals appearing as doublets in positions C-4 and
C-6 was observed.
Figure 1. UV/Vis spectra of 3-HAA and 3-HOA oxidation media.
Reaction media contained substrate (1 m), laccase (100 U L–1)
and phosphate (100 m) at pH values ranging from 2 to 10. After
24 h incubation at 25 °C, the spectra of 10-fold diluted solutions
were recorded.
Biotransformation of 3-HAA and 3-HOA by Use of Fungal
Laccase – HPLC Analysis
3-HAA and 3-HOA (1 m) were subjected to laccase
(100 U L–1) oxidation in phosphate solution (0.1 ) over
24 h at 25 °C, at pH values ranging from 2 to 10 (Scheme 1).
The HPLC method used for 3-HAA oxidation products
The biotransformations were followed by UV spectroscopy analysis was inspired by Iwahashi et al.[32] The biotransfor-
(Figure 1). With both substrates, appearance of colour was mation media showed the appearance of cinnabarinic acid
rapidly observed at pH3–5. Indeed, the commercial laccase (CA) and several by-products, amounts of which increased
Oxizym LA displays its maximum activity towards a refer- when reactions were performed at low pH. The identifica-
ence compound – namely 2,2Ј-azino-bis(3-ethylbenzthiaz- tion of CA as the oxidation product through different pro-
oline-6-sulfonic acid) (ABTS) – at pH3 (data not shown). cesses had already been studied.[33–35] We confirmed that
Media containing 3-HAA became progressively orange/ the main product of 3-HAA oxidation by laccase is CA by
brown between pH3 and 7, while media containing 3-HOA comparison of the UV/Vis scan with data reported by Eg-
became bright yellow under the same conditions. After 24 h gert et al.[2] and by mass spectrometry analysis (APCI, posi-
incubation, maximum colouration was obtained at pH6–7. tive mode), which showed the expected peak [M + H] at
It can be noted that at lower pH, the colour of the media 301 m/z, in accordance with previous literature results.[2,3,32]
did not evolve significantly after 15 h. It seems that the re-
Analysis of 3-HOA biotransformation media required an
placement of the carboxylic acid function by the sulfonic appropriate method to be set up. Separation of organic sul-
acid one did not drastically affect the substrate reactivity. fonic acids by HPLC is not a trivial issue.[36,37] Our optimal
Media containing 3-HAA showed a light coloration upon analytical conditions allowed the elution of the substrate
storage at pH8 to 10, even in the absence of laccase. This after a few minutes, while the oxidized products were de-
phenomenon is probably due to substrate autooxidation.[32] tected over the 40 min run. All the oxidized products were
Such a reaction was less visible in the case of 3-HOA. The detected at 220/254 nm, and the orange-red coloured com-
shape of the UV/Vis spectrum of 3-HAA oxidation medium pounds were also monitored at 440/460 nm (Table 2, en-
around pH6–7 strongly resembled that of pure cinnabarinic tries 1–5).
acid (CA),[2] while spectra recorded at lower pH differed
HPLC analyses showed the appearance of at least three
significantly. Similarly, with 3-HOA, UV/Vis spectra shape orange-coloured compounds in the crude media at reten-
of the media seemed to indicate that several by-products tion times (tR) of 2.1, 23 and 26 min. As observed in the
are present at lower pH. This suggests that more neutral case of 3-HAA, the numbers and amounts of by-products
conditions favour the appearance of the highly coloured were higher at lower pH. At pH6 no residual substrate
phenoxazinone product; this was checked more accurately could be detected, and the compound eluting at 23.3 min
through HPLC analyses.
was produced with a conversion ratio of about 85%.
Eur. J. Org. Chem. 2008, 72–79
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75

J. Marchand-Brynaert et al.
FULL PAPER
Table 2. HPLC studies of biotransformation reactions.[a]
analysis of which indicated a purity of about 65% in LAO.
A portion of this residue was then subjected to chromatog-
raphy on reversed-phase silica RP-18 with water as solvent.
The major red fraction was collected again and analysed by
HPLC. LAO was isolated with a purity of about 80%
(Table 2, entry 10). However, another unidentified oxidized
Retention time tR [min][b] and ratio of peak area (%)[c]
Entry pH Buffer 3-HOA By-products
LAO
By-products
tR = 1.9
2ϽtR Ͻ24 tR = 23–24
tR Ͼ25
1[d]
2[d]
3[d]
4[d]
5[d]
6[e]
3
4
5
6
7
4
5
6
6
6
6
yes
yes
yes
yes
yes
no
no
no
no
no
no
2%
2%
–
–
–
10%
–
–
–
–
25%
30%
15%
6%
25%
55%
15%
2%
–
7%
50%
40%
10%
10%
20%
20%
20%
25%
20%
9%
75%
85%
40%
10%
48%
70%
65%
85%
90%
product with a red colouration was still present (tR
=
26 min). Finally, preparative HPLC followed by filtration
through a short pad of reversed-phase for removal of the
residual MSA allowed the recovery of LAO with a purity
of about 90% (Table 2, entry 11). The ESI mass spectrum
of purified LAO showed a peak at 371 m/z with 100% in-
tensity corresponding to the structure 4 (Scheme 1) minus
one atom of hydrogen. Adducts of sodium and potassium
7[e]
8[e]
9[f]
13%
3%
–
10[g]
11[h]
–
7%
[a] The elution system for the analysis of 3-HOA and its oxidation
products consisted of: A (H2O/MSA, 100:0.1, v/v), B (H2O/
CH3CN/MSA, 75:25:0.01, v/v/v), C (H2O). After a 5 min isocratic
step at 100% A, a linear gradient to 100% C over 5 min was ap-
plied, followed by a second gradient to 100% B over 15 min. A
10 min isocratic step at 100% B was applied to ensure the elution
of all oxidation products, online UV scans were performed, samples
were taken after 24 h. [b] Retention times obtained as triplicates.
[c] %Area of peaks detected at 235 nm. [d] Sample diluted at
0.5 m with MSA/H2O. [e] Sample at 5 m, directly taken from
crude reaction mixture. [f] Sample of 5 m concentration from
crude medium of large-scale biotransformation. [g] Sample at 5 m
of fraction collected after chromatography on RP18. [h] Sample at
5 m of fraction collected after preparative HPLC.
1
could also be detected in lower proportions. H NMR in
DMSO showed the two hydrogen atoms of the amino group
Use of preparative HPLC was envisaged for purifying
this main oxidation product. The presence of phosphate
salts from the buffer caused problems when concentration
of the crude reaction mixtures was necessary, so the laccase
oxidation reaction of 3-HOA was therefore assayed in ab-
sence of buffer, the pH being adjusted to pH4, 5 and 6 by
addition of NaOH/HCl. The colouration of the unbuffered
media followed almost the same kinetics as that of the buff-
ered ones, and no significant pH modification was observed
during the biotransformations. HPLC analyses confirmed
that the same products were obtained in almost similar
amounts (Table 2, entries 6–8). The UV/Vis spectrum of the
major red compound at room temp. and 23.3 min was sim-
ilar to that of a 2-aminophenoxazinone.[2,3] Its mass spec-
trum indicated the presence of a dimeric structure.
Figure 2. HPLC chromatogram of bioconversion reaction with
commercial laccase Bioscreen (Trametes versicolor) at 25 °C with
substrate 3-HOA (5 m). pH6 (adjusted with HCl/NaOH), sample
taken after 24 h of reaction. LAO, major red compound of interest.
Elution conditions from Table 2.
Table 3. NMR spectroscopic data (δ values, ppm) for 2-amino-3-
oxo-3H-phenoxazin-1,9-disulfonic acid (4, LAO).
H
δ[a]
δ[b]
C atom δ[c]
atom
Identification of the Main Product Obtained Through 3-
HOA Oxidation by Laccase
–
2
–
4
–
–
7
8
–
–
–
–
1
2
3
4
5
6
7
8
123.2
139
176.3
105
150.1
141.6
117
9.87 (brs), 9.45 (brs)
–
6.65 (s)
–
–
A large-scale biotransformation was conducted at a
higher concentration of substrate (5 m) and enzyme
(200 U L–1) at pH6 in phosphate-free medium. The reac-
tion was stopped after 24 h by decreasing the pH with MSA
in order to inactivate the laccase.
6.56 (s)
–
–
7.76 (d), J = 8 Hz 7.74 (d), J = 7.5 Hz
7.43 (t), J = 8 Hz 7.5 (dd), J = 7.5 Hz;
6.9 Hz
127
The ratio of substrate conversion was determined by ana-
lytical HPLC of the crude mixture (Figure 2). Detection at
220/254 nm clearly showed the complete consumption of 3-
HOA. The major red compound, named LAO (which
stands for laccase acid orange), was obtained efficiently and
the proportion of by-products was rather low (Table 2, en-
9
–
–
–
7.57 (d), J = 8 Hz 7.58 (d), J = 6.9 Hz
9
123.8
136.8
118.3
153.7
–
–
–
–
–
–
10
11
12
[a] 500 MHz, D2O. [b] 300 MHz, DMSO/CD3OD (95:5, v/v).
[c] 75 MHz DMSO/CD3OD (95:5, v/v), D1 = 5 s; for atoms num-
try 9). Freeze drying gave a residual red powder, HPLC bering, see Figure 2.
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Novel Phenoxazinone Dye
1
5 s). Low-resolution mass spectra were acquired with a Thermo
Finnigan LCQ spectrometer, either in positive mode APCI, or
negative mode ESI or EI methods (70 eV). High-Resolution Mass
Spectrometry (HRMS) analysis by the ESI method was performed
at the University of Mons Hainaut (Belgium). Melting points were
recorded with an Electrothermal apparatus calibrated with benzoic
acid. Infrared spectra were recorded with a Shimadzu FT-IR 84005
spectrometer with KBr plates. The HPLC system consisted of
Waters pumps and a Waters 996 photodiode array detector (for
analytic separations) or a Waters 486 absorbance detector (for
as nonequivalents at 9.9–9.4 ppm. H NMR in D2O gave
the proton signals corresponding to the expected core for
the sulfonic analogue of cinnabarinic acid. 13C NMR
clearly showed the twelve carbon atoms of phenoxazinone 4
(Table 3). 2D correlation experiments (HMQC and HMBC)
provided unambiguous structural assignment of the novel
dye LAO. The J3 coupling observed by HMBC established
a correlation between the proton H-8 and the two carbons
C-10 and C-6. Similar correlations could be detected for the
protons H-7 and H-9 and the carbons C-9/C-11 and C-7/ semipreparative HPLC) (Waters, Milford, Massachusetts, USA).
Analytical separations were performed with Waters XterraMSC18
column (4.6ϫ100 mm, 5 µm) (Waters, Milford, Massachusetts,
USA) fitted with a conventional pre-column. Detection was per-
formed at 220/254 nm with control at 440/460 nm, and on-line UV/
Vis absorbance scans were performed. Semipreparative separation
was performed with a Waters Xterra-PrepMSC18 OBD column
(30ϫ100 mm, 5 µm) (Waters, Milford, Massachusetts, USA) fitted
with a conventional pre-column. Detection was performed at
300 nm. All the solvents were of HPLC grade. Flow rate was
1 mLmin–1 for analytical separation and 15 mL min–1 for semipre-
parative separation.
C-11, respectively. Finally, a correlation between the proton
H-4 and the two carbons C-12 and C-2 allowed the com-
plete characterization of LAO.
Conclusions
We have disclosed the first efficient synthesis of 3-hy-
droxyorthanilic acid (3-HOA), in five steps from 2-ami-
nophenol, with an overall yield of 50%. This compound
had previously been reported only as a by-product, and had
not been fully characterized. Our process, making use of the
selective ortho-metallation reaction to instal the sulfonate
group, was feasible on multi-gram scales. Moreover, the
study of O/N deprotection conditions for intermediate 9 led
to the discovery of a novel benzoxazole derivative 11, which
will be further exploited for the preparation of tetrasubsti-
tuted benzenesulfonic acids. Regarding 3-HOA as a good
potential substrate for laccase, similarly to 3-HAA, we were
able to prepare a new red dye of phenoxazinone type (LAO)
resulting from an oxidative dimerization of the precursor.
This biotransformation mimics the well known synthesis of
cinnabarinic acid (CA). Our study has established the best
experimental conditions (pH, concentrations, ...) for pro-
ducing high yields of LAO, with a minimum amount of
side-products, as well as analytical and preparative condi-
tions for HPLC analysis and purification of sulfonated
products.
The mechanism of laccase oxidation is still under investi-
gation. For instance, the increased reactivity of aminophe-
nol derivatives at acidic pH values suggests that their pro-
tonation state could modify the relative oxidation potentials
of aromatic amine and hydroxy functions.[38] The primary
oxidation product would be a protonated arylimino radical
rather than the protonated aryloxy radical usually obtained
in neutral solutions.
The new phenoxazinone derivative (LAO) has interesting
colour properties: it has a high molar extinction coefficient
at 450 nm and is totally water-soluble, as required for bio-
logical detection systems.
Organic Synthesis: For standard working practice, see recent publi-
cation.[40] Reactions with organolithium reagents were carried out
under argon in flame-dried glassware. Anhydrous tetrahydrofuran
and dichloromethane were purchased from Fluka, and chemical
reagents and reactants were from Aldrich and Acros. Thin layer
chromatography was carried out on aluminium-backed silica gel
plate F254 (Merck) and visualized by use of UV irradiation or
iodine vapour. Column chromatography was carried out either with
flash silica gel (60–200 µm) or with silica gel 60-RP18 (40–60 µm)
as the stationary phase with elution by the flash chromatography
technique.
Biotransformation: Laccase from Trametes versicolor was purchased
as a brownish powder from Bioscreen e. K. (Uebach-Palenberg,
Germany) under the commercial trademark Oxizym LA (batch
no. LA 2000/001). Laccase activity was determined by oxidation of
ABTS [2Ј,2-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid)]
(4 m, Sigma–Aldrich) into a stable cationic radical ABTS·+ (ab-
sorption coefficient, εM = 34220 –1 cm–1) in tartaric buffer
(100 m) at pH4.5 and at 25 °C. One unit was defined as the
amount of enzyme to oxidize 1 µmol of ABTS per minute. Analyti-
cal biotransformations were performed at 25 °C in solutions con-
taining 3-HAA or 3-HOA (1 m), phosphate buffer (0.1 ) and
laccase (100 U L–1), unless otherwise mentioned. All reactions were
monitored by spectrophotometry between 300 and 800 nm with a
Beckman DU-800 spectrophotometer (Analis, Namur, Belgium)
connected to a high-performance temperature controller.
N-(2-Hydroxyphenyl)-2,2-dimethylpropionamide: NaHCO3 (110.1
mmol, 9.2 g) and pivaloyl chloride (40.37 mmol, 4.9 mL) were
added to a solution of 2-aminophenol (36.7 mmol, 4 g) in ethyl
acetate/water (250/300 mL). The mixture was stirred for 4 h at
room temperature. The layers were separated and the organic layer
was washed with aqueous HCl (1 ). The organic layer was dried
with Na2SO4 and the solvents were evaporated in vacuo. The resi-
due was purified by column chromatography on flash silica gel (cy-
clohexane/ethyl acetate, 7:3) to give the title compound as a white
solid.[41] Yield 91% (6.5 g). C11H15NO2 (193.11). 1H NMR
(500 MHz, DMSO): δ = 9.79 (s, 1 H, OH), 8.54 (s, 1 H, NH), 7.76
Experimental Section
1
General Methods: H and 13C NMR spectra were recorded with a
Bruker Avance 500 or a Bruker Avance 300 spectrometer. Spectra
were obtained in [D6]DMSO, CDCl3, D2O or CD3OD. Chemical (dd, JH,H = 7.8, 1.9 Hz, 1 H, Ar–H), 6.93 (td, JH,H = 7.8, 1.9 Hz,
shifts are reported in ppm relative to the solvent signals.[39] 13C
NMR were obtained with broadband proton decoupling (D1 =
1 H, Ar–H), 6.86 (dd, JH,H = 6.8, 1.9 Hz, 1 H, Ar–H), 6.77 (td,
JH,H = 7.8, 1.9 Hz, 1 H, Ar–H), 1.23 (s, 9 H, 3ϫCH3) ppm. 13C
Eur. J. Org. Chem. 2008, 72–79
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
77

J. Marchand-Brynaert et al.
FULL PAPER
NMR (75 MHz, DMSO): δ = 176.1 (CO amide), 147.4 (C-1), 126.2
(C-2), 124.29 (C-5), 121.5 (C-4), 118.8 (C-3), 115.2 (C-6), 38.7
[C(CH3)3], 27 (3ϫCH3) ppm. EI-MS: m/z (%) = 193.3 (55) [M]+,
136 (7), 109 (15), 85 (45), 57.1 (40).
286.0749; found 286.0758. ESI-MS: m/z (%)
[M – H]–, 270.9 (25), 214 (70), 206 (40).
=
286.1 (100)
2-(2,2-Dimethylpropionylamino)-3-hydroxybenzenesulfonic
Acid
(10): BCl3 (1 , 10.78 mmol, 10.8 mL) was added dropwise to a
solution of 9 (13.5 mmol, 1.02 g) in CH2Cl2 (15 mL), kept at 0 °C.
The mixture was left 1 h at 0 °C and then overnight at 25 °C. The
reaction mixture was cooled to 0 °C, and H2O (20 mL) was added
dropwise to quench the residual BCl3. After evaporation, the resi-
due was dissolved in MeOH (6 mL) and added to RP18 (0.1 g) and
concentrated under vacuum. The residue was filtered through a
0.5 µm filter with MeOH/H2O (1:1 v/v, 60 mL), and the solution
was concentrated to yield the title compound as a brown solid.
Yield 91% (0.87 g). NOTE: The compound exists partially in the
form of benzoxazole 11 in a ratio of 70:30 in favour of product 10.
N-(2-Methoxyphenyl)-2,2-dimethylpropionamide
(5a):
K2CO3
(3.4 mmol, 472 mg) and methyl iodide (3.4 mmol, 0.21 mL) were
added to a solution of N-(2-hydroxyphenyl)-2,2-dimethylpropiona-
mide (3.1 mmol, 600 mg) in DMF (2 mL). The solution was stirred
for 24 h at 20 °C. The reaction mixture was diluted with water
(15 mL) and extracted with ethyl acetate (3ϫ15 mL). The organic
layer was dried with MgSO4 and evaporated in vacuo. The residue
was purified by column chromatography on flash silica gel (cyclo-
hexane/ethyl acetate, 9:1, 5:5, and 0:10) to give the title com-
pound.[42] Yield 94%. C12H17NO2 (207.27). 1H NMR 300 MHz
(CDCl3): δ = 8.42 (d, JH,H = 8 Hz, 1 H, Ar–H), 8.13 (s, NH), 7.03
(t, JH,H = 7.8 Hz, 1 H, Ar–H), 6.95 (t, JH,H = 6.8 Hz, 1 H, Ar–H),
6.87 (d, JH,H = 7.8 Hz, 1 H, Ar–H), 3.89 (s, 3 H, OCH3), 1.31 (s,
9 H, 3ϫCH3) ppm. 13C NMR (75 MHz, CDCl3): δ = 176.4 (CO
amide), 147.9 (C-1), 127.8 (C-2), 123.3 (C-5), 121.07 (C-4), 119.4
1
C11H15NO5S (273.3055). H NMR (300 MHz, CD3OD): δ = 7.52
(d, JH,H = 6.8 Hz, 1 H, Ar–H), 7.16 (t, JH,H = 7.8 Hz, 1 H, Ar–
H), 7.05 (d, JH,H = 6.8 Hz, 1 H, Ar–H), 3.68 (s, 0.5 H), 1.36 (s, 9
H, 3ϫCH3) ppm.
2-tert-Butylbenzoxazole-4-sulfonic Acid (11): TFA (1.8 mmol,
(C-3), 109.7 (C-6), 55.7 (O–CH3), 39.9 [C(CH3)3], 27.5 0.2 mL) was added to a solution of a 10+11 mixture as a dry pow-
(3ϫCH3) ppm. EI-MS: m/z (%) = 207.1 (100) [M]+, 176 (10), 164
(7), 150 (15), 123 (45), 107.9 (40), 57.1 (48).
der (3.6 mmol, 1 g) in MeOH (10 mL). The mixture was stirred for
3 h at 80 °C. The reaction mixture was then cooled to 25 °C and
evaporated under vacuo to yield the title compound as a pale yel-
low solid; yield 99% (0.92 g). C11H13NO4S (255). M.p. 293–295 °C.
1H NMR (300 MHz, CD3OD): δ = 7.86 (d, JH,H = 7.5 Hz, 1 H,
Ar–H), 7.77 (d, JH,H = 7.5 Hz, 1 H, Ar–H), 7.48 (t, JH,H = 7.8 Hz,
1 H, Ar–H), 1.56 (s, 9 H, 3ϫCH3) ppm. 13C NMR (125 MHz,
CD3OD/CDCl3): δ = 176.8 (C-2), 151.1 (C-1), 134.3 (C-4), 125.3
(C-6), 124.7 (C-3), 113.3 (C-5, C-7), 35.8 [C(CH3)3], 20.5
2-(2,2-Dimethylpropionylamino)-3-methoxybenzenesulfonic Acid (9):
n-Butyllithium in cyclohexane (2.5 , 40.5 mmol, 16.2 mL) was
added dropwise under argon at –10 °C to a solution of 5a
(19.3 mmol, 4 g) in anhydrous THF (40 mL). The solution was kept
for 15 min at low temperature (between –10 °C and 0 °C), then
warmed to 25 °C and kept for 3 h at room temperature. The di-
anion solution was added dropwise at – 10 °C to a stirred suspen-
sion of crystalline sulfur trioxide/trimethylamine complex (powder
dried prior to use under vacuum for 1 h, 28.95 mmol, 4.028 g) in
anhydrous THF (10 mL). The reaction mixture was allowed to re-
act for 2 h at low temperature (between –10 °C and 0 °C; white-
orange suspension) and was then warmed up to room temperature
for 20 h (orange-brown solution). Aqueous NaOH (10% w/w,
150 mL) was then added and the mixture was extracted with diethyl
ether (3ϫ100 mL) to remove residual starting material. Tetrabu-
tylammonium hydrogen sulfate (17.37 mmol, 5.89 g) was added to
the aqueous solution, and the mixture was extracted with ethyl ace-
tate (3ϫ100 mL). The combined organic phases were dried with
MgSO4 and evaporated (black oil). This residue was purified on
anion-exchange resin IRA-900 (chloride form) with MeOH/H2O
(1:1, v/v) as neutral eluent and MeOH/1  HCl (1:1, v/v) as acidic
eluent. The IRA mass needed was 100 g. First the resin was placed
in H2O (200 mL) and allowed to swell for 24 h, and then the resin
was placed in a column (diameter 2 cm) and washed with HCl (1 ,
150 mL), followed by 150 mL of MeOH/HCl 1  (1:1, v/v). The
crude salt 8, dissolved in the minimum possible amount of MeOH,
was added dropwise onto the resin. The resin was allowed to stand
undisturbed for 30 min after complete percolation of the mixture
into the resin. Afterward the resin was washed with neutral eluent
(500 mL) to recover the tetrabutylammonium chloride salt. The
resin was then washed with of acidic eluent (500 mL) and the solu-
tion was concentrated under vacuo to yield the title compound as
a brown gummy solid. Yield 70% (3.9 g). C12H17NO5S (287). 1H
NMR (300 MHz, CD3OD): δ = 7.48 (dd, JH,H = 7.8, 1.5 Hz, 1 H,
(3ϫCH ) ppm. IR (KBr): ν = 3448, 2977, 2360, 1566, 1473, 1427,
˜
3
1253, 1191, 1141, 1045, 891, 802, 682 cm–1. HRMS-ESI negative
mode: m/z calcd: 278.0463 for C11H12NO4NaS, found 278.0466.
ESI-MS: m/z (%) = 254.2 [M – H] (100), 190.2 (15).
2-Amino-3-hydroxybenzenesulfonic Acid (3, 3-HOA). Method i:
Compound 11 (3.1 mmol, 0.8 g) was added to a solution of HCl
(6 , 21 mL). The mixture was heated at 80 °C for 24 h. Then the
solution was cooled down and the solvents were evaporated. The
residue was dissolved in MeOH (20 mL), and activated charcoal
was added (50 mg). The mixture was stirred at 40 °C for 30 min.
The solution was then filtered through a short pad of celite and
the pad was washed with hot methanol (30 mL). The filtered solu-
tion was concentrated and dried under vacuo to yield the title com-
pound as a light brown solid. Yield 90% (0.53 g). The purity of the
compound can be monitored by HPLC (tR = 1.8 min). C6H7NO4S
(189.1). M.p. 210–215 °C (slow dec.). 1H NMR (300 MHz,
CD3OD): δ = 7.41 (dd, JH,H = 7.8, 1.35 Hz,1 H, Ar–H), 7.34 (t,
JH,H = 8.1 Hz, 1 H, Ar–H), 7.07 (dd, JH,H = 7.8, 1.35 Hz, 1 H, Ar–
H) ppm. 13C NMR (125 MHz, CD3OD): δ = 152.6 (C-3), 140.6 (C-
1), 130.6 (C-5), 119.4 (C-6), 118.8 (C-4), 115.8 (C-2) ppm. IR
(KBr): ν = 3487, 3436, 3220, 3066, 2931, 2692, 1635, 1490, 1469,
˜
1365, 1307, 1191, 1064, 1037, 902, 798, 675 cm–1. HRMS-ESI nega-
tive mode: m/z calcd. 188.0018 for C6H6NO4S; found 188.0018.
ESI-MS: m/z (%) = 188.07 [M – H] (100), 376.8 [2 M – 2 H] (75),
187.1 (6), 108 (5), 79.8 (15).
2-Amino-3-hydroxybenzenesulfonic Acid (3, 3-HOA). Method j:
Compound 9 (1.04 mmol, 0.3 g) was added to a solution of HBr
Ar–H), 7.30 (t, JH,H = 8.4 Hz, 1 H, Ar–H), 7.17 (dd, JH,H = 7.8, (48%, 6 mL). The mixture was heated at 120 °C for 13 h. After-
1.5 Hz, 1 H, Ar–H), 3.83 (s, 3 H, OCH3), 1.33 (s, 9 H,
wards the reaction mixture was cooled down. Part of the product
was recovered as a solid by precipitation in situ. The filtered solu-
3ϫCH3) ppm. 13C NMR (125 MHz, CD3OD): δ = 179.5 (CO
amide), 156.4 (C-3), 141.8 (C-1), 127.9 (C-5), 124.1 (C-2), 119.7 (C- tion was concentrated, and the residue was dissolved in MeOH
6), 115.5 (C-4), 56.5 (OCH3), 39.9 [C(CH3)3], 27.6 (3ϫCH3) ppm. (20 mL). Activated charcoal was added (50 mg), and the mixture
HRMS-ESI negative mode: m/z [M – H]– calcd. for C12H16NO5S:
was agitated at 40 °C for 30 min. The solution was then filtered
78
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 72–79

Novel Phenoxazinone Dye
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through a short pad of celite, and the celite was washed with hot
methanol (30 mL). The filtered solution was concentrated and
dried under vacuum to yield the title compound as a brown solid.
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[13]
[14]
[15]
[16]
[17]
2-Amino-3-oxo-3H-phenoxazin-1,9-disulfonic Acid (4, LAO):
A
solution of laccase (200 U L–1) and 3-HOA (5.29 mmol, 1.02 g) in
water (final volume of 100 mL) adjusted to pH6 with HCl and
NaOH was prepared. The stirred mixture was kept for 24 h at
25 °C. The reaction was stopped by decreasing the pH with MSA
(pH ≈ 2). The crude mixture was freeze-dried to give a red powder
(1 g). Part of this powder (400 mg) was purified by chromatography
on reversed phase (water as eluent) to yield an almost pure fraction
of LAO (270 mg). Part of this fraction (70 mg) was subjected to
preparative HPLC to yield the pure title compound as a deep red
solid (30 mg). Yield of about 70% determined by HPLC.
C12H8N2O8S2 (372.3305). 1H NMR and 13C NMR: see Table 3.
[18]
[19]
[20]
M.p. Ͼ300 °C. IR (KBr): ν = 3433, 2364, 1635, 1593, 1473, 1415,
˜
1218, 1053 cm–1. UV: λmax at 435 and 339 nm (0.1  phosphate
buffer, pH7). 435 nm (ε = 13981 Lmol–1 cm–1). UV: λmax at 460 and
330 nm (0.1  phosphate buffer, pH2). 460 nm (ε
=
[21]
5426 Lmol–1 cm–1). HRMS-ESI negative mode: m/z calcd.
370.9644 for C12H7N2O8S2; found 370.9641. ESI-MS: m/z (%) =
371.16 [M – H] (100); 393.1 [M – H + Na] (40); 291.2 (20).
[22]
[23]
[24]
Supporting Information (see also the footnote on the first page of
this article): 1H and 13C NMR spectra of compounds 9 (Figures
S2, S3), 11 (Figures S4, S5), 3 (Figures S6, S7), 4 (Figures S8, S9)
are provided as additional material. UV/Vis spectra of compound
4 at pH7, pH2 (Figures S10) are also furnished.
[25]
[26]
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[28]
Acknowledgments
[29]
[30]
This research was supported by the European Commission, Sixth
Framework Program (NMP2-CT2004-505899, SOPHIED).
T.-V. T., M. I. and D. I. are gratefully acknowledged for their tech-
nical assistance. J. M.-B. is senior research associate of the Belgian
FRNS (Fonds National de la Recherche Scientifique, Belgium).
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Received: September 12, 2007
[40]
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[42]
Published Online: November 27, 2007
Eur. J. Org. Chem. 2008, 72–79
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
79