Investigation of the mechanism of action of pyrogallol-phloroglucinol transhydroxylase by using putative intermediates

Article abstract of DOI:10.1002/chem.200601053

Pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici, a molybdopterin-containing enzyme, catalyzes a key reaction in the anaerobic degradation of aromatic compounds. In vitro, the enzymatic reaction requires 1,2,3,5-tetrahydroxy-benzene as a cocatalyst and the trans-hydroxylation occurs without exchange with hydroxy groups from water. To test our previous proposal that the transfer of the hydroxy group occurs via 2,4,6,3′,4′, 5′-hexahydroxydiphenyl ether as an intermediate, we synthesized this compound and investigated its properties. We also describe the synthesis and characterization of 3,4,5,3′,4′,5′-hexahydroxydiphenyl ether. Both compounds could substitute for the cocatalyst in vitro. This indicates that the diphenyl ethers can intrude into the active site and initiate the catalytic cycle. Recently, the X-ray crystal structure of the transhydroxylase (TH) was published^[16] and it supports the proposed mechanism of hydroxy-group transfer.

Full text of DOI:10.1002/chem.200601053

FULL PAPER
DOI: 10.1002/chem.200601053
Investigation of the Mechanism of Action of Pyrogallol–Phloroglucinol
Transhydroxylase by Using Putative Intermediates
Csaba Paizs,[a, b] Ulrike Bartlewski-Hof,[a] and Jµnos RØtey*[a]
Dedicated to Professor Bernhard Kräutler on the occasion of his 60th birthday
Abstract:
transhydroxylase from Pelobacter acidi-
gallici, molybdopterin-containing
Pyrogallol–phloroglucinol
transfer of the hydroxy group occurs
via 2,4,6,3’,4’,5’-hexahydroxydiphenyl
ether as an intermediate, we synthe-
sized this compound and investigated
its properties. We also describe the syn-
3,4,5,3’,4’,5’-hexahydroxydiphenyl
ether. Both compounds could substi-
tute for the cocatalyst in vitro. This in-
dicates that the diphenyl ethers can in-
trude into the active site and initiate
the catalytic cycle. Recently, the X-ray
crystal structure of the transhydroxy-
lase (TH) was published[16] and it sup-
ports the proposed mechanism of hy-
droxy-group transfer.
a
enzyme, catalyzes a key reaction in the
anaerobic degradation of aromatic
compounds. In vitro, the enzymatic re-
action requires 1,2,3,5-tetrahydroxy-
benzene as a cocatalyst and the trans-
hydroxylation occurs without exchange
with hydroxy groups from water. To
test our previous proposal that the
thesis
and
characterization
of
Keywords: enzymes
molybdopterin (Moco)
transfer · reaction mechanisms
·
ethers
·
·
OH
Introduction
droxy groups are incorporated into the product from
water.[5] On the basis of these results, the reaction was for-
mulated[4,5] as depicted in Scheme 1. The 2-OH group from
1,2,3,5-tetrahydroxybenzene is transferred to pyrogallol,
whereby phloroglucinol and a new molecule of the cocata-
lyst are produced. Such ingenious use of a cocatalyst already
has precedence in enzyme chemistry; for example, the gly-
colytic enzymes, phosphoglucomutase and phosphoglycerate
mutase, take advantage of their cocatalysts, that is, glucose-
1,6-bisphosphate and glycerate-2,3-bisphosphate, respective-
Pyrogallol–phloroglucinol transhydroxylase (TH) from Pelo-
bacter acidigallici is one of the key enzymes in the anaerobic
degradation of aromatic compounds like gallic acid and vari-
ous phenols.[1–3] All these compounds are converted into
phloroglucinol, which is then reductively dearomatized and
degraded to three acetyl-coenzyme A (acetyl-CoA) mole-
cules via 3-hydroxy-5-oxohexanoate. This pathway leads not
only to important building blocks but can also be used for
adenosine triphosphate (ATP) synthesis.
Schink and co-workers showed that TH contains a molyb-
dopterin cofactor (Moco) and iron sulphur clusters [4Fe–4S]
and needs 1,2,3,5-tetrahydroxybenzene as a cosubstrate, or
rather as a cocatalyst because it is regenerated in the reac-
tion cycle.[1–6] Furthermore, as shown by 18O labeling, no hy-
[a] Dr. Cs. Paizs, U. Bartlewski-Hof, Prof. Dr. J. RØtey
Institute of Organic Chemistry and Biochemistry
University of Karlsruhe
Richard-Willstätter-Allee, 76128 Karlsruhe (Germany)
Fax : (+49)721-608-4823
E-mail: janos.retey@ioc.uka.de
[b] Dr. Cs. Paizs
Department of Biochemistry and Biochemical Engineering
Babes-Bolyai University
Arany Jµnos str. 11, 400028 Cluj-Napoca (Kolozsvµr) (Romania)
Scheme 1. The role of 1,2,3,5-tetrahydroxybenzene in the mechanism of
action of the Mo-cofactor-containing pyrogallol–phloroglucinol transhy-
droxylase as postulated by Schink and co-workers.[4,5]
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3,4,5,3’,4’,5’-hexahydroxydi-
phenyl ether (6 and 7, respec-
tively) and their interaction
with the transhydroxylase.
Results and Discussion
Synthesis of hexahydroxydi-
phenyl ethers: 2,4,6,3’,4’,5’- and
3,4,5,3’,4’,5’-Hexahydroxydi-
phenyl ethers (6 and 7, respec-
tively) were found as compo-
nents of the ethyl acetate solu-
ble mixture of polyhydroxy-
phenyl esters occurring in vari-
ous kinds of algae, such as
Bifurcaria bifurcate and Capro-
phyllum
maschalocarpu-
mas.[11,12] Only the hexaacetylat-
ed form of the diphenyl ether
was isolated; the unprotected
compound could not be ob-
tained in pure form due to its
oxygen sensitivity. Both 6 and 7
have previously been synthe-
sized from the corresponding
Scheme 2. Proposed mechanism for the action of the pyrogallol–phloroglucinol transhydroxylase. Oxidation of
pyrogallol to the corresponding orthoquinone allows nucleophilic attack by the 2-OH group of the tetrahy-
droxybenzene cocatalyst.[9]
ly. In these cases, the substrates are converted into the coca-
talysts by phosphate transfer, while the original cocatalyst
becomes the product.[7] In the formulation of a detailed
mechanism for the TH reaction, the mode of hydroxy trans-
fer is a matter of debate. It is believed that in Moco-contain-
ing hydroxylases which use water as the hydroxy group
source, the OH group is first ligated to the molybdenum and
then transferred to the substrate; a similar mechanism was
also discussed for the TH reaction.[8]
hexamethoxydiphenyl ethers by using boron tribromide as a
demethylation agent,[13] but the products were not isolated
and were transformed into dibenzofurans.[14] The syntheses
of 6, 7 and 1,2,3,5-tetrahydroxybenzene (1) are presented in
Schemes 3 and 4. Using modern HPLC methods, we success-
fully purified these compounds. The hexahydroxydiphenyl
ethers were characterized for the first time by spectroscopic
methods (1H and 13C NMR spectroscopy and mass spec-
trometry). They had to be handled under an inert gas atmos-
phere.
A second mechanistic proposal by RØtey and co-workers[9]
(see also reference [10]) re-
quires direct hydroxy transfer
from the cocatalyst 1,2,3,5-tet-
rahydroxybenzene to the ortho-
quinone form of pyrogallol.
Here, the main function of
Moco is the oxidation of pyro-
gallol to quinone. Along the
postulated pathway, an enzyme-
bound 2,4,6,3’,4’,5’-hexahydroxy-
diphenyl ether is generated as
an intermediate (Scheme 2).
Fragmentation of this diphenyl
ether into phloroglucinol and
the orthoquinone form of tetra-
hydroxybenzene is followed by
reduction of the latter to regen-
erate the cocatalyst. Here we
describe the synthesis of
Scheme 3. Synthesis of the proposed intermediate, 2,4,5,3’,4’,6’-hexahydroxydiphenyl ether (6). mCPBA=
2,4,6,3’,4’,5’-hexahydroxy- and meta-chloroperbenzoic acid.
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Transhydroxylase Mechanism
FULL PAPER
When pure transhydroxylase,
isolated as described by Schink
and co-workers,[5] was incubat-
ed with pyrogallol only, no re-
action occurred as monitored
by HPLC. In the presence of 1,
phloroglucinol was produced;
after
a
longer incubation
(15 min) at 308C, the concen-
tration of 1 was only slightly de-
creased (Figure 2), as it was re-
generated in the reaction cycle.
This observation is in good
agreement with the proposed
role of 1 as the cocatalyst. The
enzyme assay is a modification
of that described by Schink and
co-workers.[4–6] Based on the
areas under the HPLC traces
and the amounts of the pyrogal-
lol (10 mmol) and enzyme
(0.1 mg) used in the reaction,
the enzyme activity can be esti-
mated to be 2 mmolminÀ1 mgÀ1
enzyme. As some oxidation of
the extremely oxygen-sensitive
polyhydroxyphenols was un-
avoidable, the limit of error is
estimated to be about 10%. To
investigate whether the transhy-
droxylase will accept 6 as a co-
catalyst or intermediate, the
latter compound and pyrogallol
were added to the enzymatic
reaction mixture (see the Ex-
Scheme 4. Synthesis of 1,2,3,5-tetrahydroxybenzene and symmetrical 3,4,5,3’,4’,5’-hexahydroxydiphenyl ether
(7).
Figure 1. Elution diagram of 1,2,3,5-tetrahydroxybenzene, pyrogallol, phloroglucinol, and the unsymmetrical
and symmetrical hexahydroxydiphenyl ethers for reference. 1 mmole of each compound was applied to the
column.
perimental
Section
and
Figure 3). Under these condi-
tions, as predicted, phlorogluci-
nol and 1 were formed. After
Interaction of the hexahydroxydiphenyl ethers with pyrogal-
lol–phloroglucinol transhydroxylase: TH is a heterodimeric
enzyme consisting of a large a and a small b subunit with
875 and 275 amino acid residues, respectively. On the basis
of its amino acid sequence, TH was considered to be a
member of the dimethylsulfoxide reductase family.[15] More
recently, the X-ray crystal structure of TH has been publish-
ed.[16] Since recombinant TH could not be expressed in the
active form,[15] the enzyme isolated directly from Pelobacter
acidigallici was used in all experiments.
Since there is no measurable difference in the UV absorp-
tion of pyrogallol and phloroglucinol, an enzyme assay was
developed that relied on HPLC analysis. Under the condi-
tions used, the retention times for authentic 1, 6, phloroglu-
cinol, pyrogallol, and 7 were approximately 4.4, 7.1, 7.9, 8.7,
and 19.3 min, respectively (Figure 1). In some cases, small
deviations from these values were observed.
60 min of reaction time, about 90% of the pyrogallol and 6
had been converted into phloroglucinol and the postulated
paraquinone form of 1,2,3,5-tetrahydroxybenzene. The latter
is supposed to remain enzyme bound and will be reduced by
pyrogallol and serve as the cocatalyst, as depicted in
Scheme 2 (Note that usually no intermediates are seen in
the HPLC analyses, except when they are added to the reac-
tion mixture; however, 1 can leak out from the active site of
the enzyme). On the basis of the areas under the HPLC
traces and the amounts of the pyrogallol, hexahydroxydi-
phenyl ether, and enzyme, it is estimated that in the enzy-
matic reaction about 2 mmol of 6 and pyrogallol were con-
sumed per minute. In other words, the kinetic competence
of this hexahydroxydiphenyl ether as an intermediate in the
reaction is shown.
In a further experiment, 6 was incubated with transhy-
droxylase. In this case no reaction occurred, but when an
equimolar amount of glutathione was introduced into the
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J. RØtey et al.
After 4 h, more phloroglucinol
was formed and the amount of
pyrogallol was reduced by
about 90% (Figure 5), while
only a relatively small portion
of 7 was consumed. According-
ly, only a very small amount of
1 can be seen in Figure 5 (peaks
between 4–5 min). The low con-
centration of the cocatalyst may
be the reason for the sluggish
reaction in this experiment. The
reactions illustrated in Figures 4
and 5 were also performed on a
preparative scale; the isolated
products showed the same ana-
lytical data as the reference 1
and phloroglucinol. These re-
sults suggest that the symmetri-
cal hexahydroxydiphenyl ether
can also intrude into the active
site of the enzyme and is
cleaved into pyrogallol and the
paraquinone form of 1, al-
though much more slowly than
its unsymmetrical counterpart
(Scheme 5). In other words, the
Figure 2. Elution diagram of the enzyme assay with pyrogallol and 1,2,3,5-tetrahydroxybenzene; after 5 min=
blue trace, after 15 min=red trace. For the conditions of the assay, see the Experimental Section.
symmetrical
hexahydroxydi-
phenyl ether does not have the
kinetic competence to be a gen-
uine intermediate in the reac-
tion.
Thus, our results support the
intermediacy of 6 in the TH re-
action and, consequently, the
mechanism previously proposed
by us (Scheme 2). At least in
this case, the formerly postulat-
ed role of Moco as an OH-
transfer agent must be revised.
Therefore, the two alternative
Figure 3. Elution diagram showing the conversion of pyrogallol into phloroglucinol and 1,2,3,5-tetrahydroxy-
benzene by transhydroxylase in the presence of 2,4,6,3’,4’,5’-hexahydroxydiphenylether; after 15 min=blue
trace, after 1 h=red trace.
enzymatic reaction mixture, phloroglucinol and 1 were
formed (Figure 4). These results show that a reducing agent
is necessary to keep the reaction going, as according to
Scheme 2 the cleavage of the hexahydroxydiphenyl ether
leads, in addition to phloroglucinol, to the enzyme-bound
paraquinone form of 1. In the absence of pyrogallol, gluta-
thione can reduce the enzyme-bound quinone to provide
the cocatalyst.
mechanisms considered by Hille et al.[9] are practically ruled
out.
The recently published X-ray crystal structure of the TH–
1,2,4-trihydroxybenzene complex[16] is consistent with the
mechanism discussed here (Scheme 2). It is now possible to
speculate about the catalytic roles of some amino acid resi-
dues found at the active site (Asp174, His144, Tyr404;
Scheme 6).[16] The 1-OH group of pyrogallol coordinates to
the MoVI atom of Moco (stage a) and is oxidized to the or-
thoquinone form, while His144 accepts the phenolic proton
(stage b). This oxidation may take place in two steps with
MoV and the semiquinone as intermediates. In the next step,
the 2-OH group of 1 adds to the quinone in a Michael-type
reaction, which is assisted by proton transfer from Asp174
(stage c). Subsequently the substrate–cocatalyst adduct is
tautomerized with the assistance of Tyr404, whereby the
The symmetrical 7 could also play the role of a cocatalyst
for TH. Its retention time (around 18 min) was, however,
completely different from that of its unsymmetrical counter-
part (Figures 1 and 5). Ether 7 alone was completely inert
with transhydroxylase and no detectable amount of phloro-
glucinol was found, even when an equimolar amount of glu-
tathione was added to the reaction mixture. Addition of py-
rogallol initiated a reaction, which produced phloroglucinol.
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Transhydroxylase Mechanism
FULL PAPER
Figure 4. Elution diagram showing the cleavage of 2,4,6,3’,4’,5’-hexahydroxydiphenyl ether catalyzed by transhydroxylase in the presence of glutathione
as a reducing agent; after 15 min=blue trace, after 1 h=red trace. The produced phloroglucinol and 1,2,3,5-tetrahydroxybenzene were isolated and iden-
1
tified by mass spectrometry and H and 13C NMR spectroscopy.
termediate (originating from
tetrahydroxybenzene) with the
assistance of Tyr404 as a proton
donor and an undefined enzy-
matic base as a proton acceptor
(stage e). The adduct is now
prepared for cleavage to phlor-
oglucinol and the paraquinone
form of the tetrahydroxyben-
zene. In this step, the previously
implied undefined enzymatic
base returns the proton to form
the product. In the last stage (f)
of the catalytic cycle, the para-
quinone form of the tetrahy-
droxybenzene is reduced by
Figure 5. Elution diagram showing the conversion of pyrogallol by transhydroxylase in the presence of
3,4,5,3’,4’,5’-hexahydroxydiphenyl ether; after 1 h=blue trace, after 4 h=red trace. The latter is consumed ac-
MoIV, thus returning the reac-
cording to the mechanism outlined in Scheme 5. The produced phloroglucinol was isolated and identified by
tion to stage b.
In conclusion, the roles as-
1
mass spectrometry and H and 13C NMR spectroscopy.
signed to the three active-site
amino acids are plausible but
remain uncertain until con-
firmed by mutation analysis.
Experimental Section
Enzymatic experiments: Transhydrox-
Scheme 5. Proposed mechanism of the cleavage of the 3,4,5,3’,4’,5’-hexahydroxydiphenyl ether (7) to 2,6-dihy-
droxyparaquinone and pyrogallol catalyzed by the transhydroxylase.
ylase was isolated as previously de-
scribed.[6] The enzyme assay was car-
ried out under anoxic conditions at
308C in
HPLC analysis of the products. All in-
gredients were stored and transferred under argon.
a discontinuous fashion by
proposed intermediate 6 is formed (stage d). The 3(5)-OH
group is still coordinated to the MoIV atom. The next step in-
volves the tautomerization of the “lower” portion of the in-
The concentrations of compounds in the assay mixture were as follows:
100 mm potassium phosphate buffer (pH 7.2), 10 mmol pyrogallol,
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J. RØtey et al.
Scheme 6. Proposed mechanism of action of the pyrogallol–phloroglucinol transhydroxylase[9] and the supposed role of certain active-site amino acid res-
idues.[16]
10 mmol 1,2,3,5-tetrahydroxybenzene, and 0.1 mg of the enzyme. The
total volume of the enzymatic assay was 1 mL. Samples (20 mL) were
withdrawn with a unimetric pipette, the reaction was terminated by
adding 0.1m H3PO4 (5 mL), and the samples were diluted 10 times with
double-distilled water before injection. The withdrawals occurred soon
after the start of the reaction and then at regular intervals. All enzymatic
reactions were carried out under anaerobic conditions. In the case of re-
actions in which pyrogallol was incubated with transhydroxylase, no re-
ducing agent was required in the enzymatic assays. Conditions for the
HPLC analyses: Nucleodur 100–5 C8 ec column with 20 mm HCl in
water/acetonitrile (95:5 v/v) as the eluent in an isocratic manner at a
1 mLminÀ1 flow rate. Chromatograms were recorded at 218 nm. The
assays with hexahydroxydiphenyl ethers were similar to the enzyme assay
described above.
spectra were recorded on a VG 7070E mass spectrometer. HPLC analy-
ses were conducted with an HP 1050 instrument.
Reagents and solvents: 2,4,6-Trimethoxybenzaldehyde, 3,4,5-trimethoxy-
aniline, 3,4,5-trimethoxyphenol, all inorganic reagents, and solvents were
from Aldrich or Fluka. All solvents were purified and dried by standard
methods as required.
Synthesis of 3,4,5-trimethoxybromobenzene (2):
A
solution of
Na2SO3·7H2O (8.57 g, 34 mmol) in water (20 mL) was added dropwise to
a stirred solution of CuSO4·5H2O (16.48 g, 66 mmol) and NaBr (10.3 g,
100 mmol) in water (60 mL). For better precipitation of CuBr, the sus-
pension was cooled to 08C. After 30 min, the supernatant was decanted
and the precipitate was washed with cold water (2”30 mL). The suspen-
sion was rendered clear by addition of concentrated HBr (26 mL) and
was stored under N2. 3,4,5-Trimethoxyaniline (9.16 g, 50 mmol) was
added to a half-concentrated HBr water solution (160 mL) in one portion
at room temperature. The suspension was heated to 708C to obtain a
clear solution, which was then cooled to 08C. Subsequently, a solution of
NaNO2 (3.45 g, 50 mmol) in water (30 mL) was added dropwise while the
temperature was kept below 58C. The reaction went to completion in
30 min and then a small portion of urea was added to destroy the un-
reacted nitrous acid. The previously prepared CuBr solution was added
to the solution of the diazonium salt at 08C under argon. The reaction
mixture was allowed to warm to room temperature and was then heated
to 1008C. The cooled mixture was extracted with diethyl ether (3”
200 mL) and the combined organic layers were washed with HCl solution
(20%, 3”100 mL), then with water (3”100 mL), and finally with KOH
For preparative purposes, an enzymatic assay with a volume of 10 mL
was used and, after the reaction was stopped with 0.1m H3PO4, the mix-
ture was heated under argon to 808C for 5 min, cooled, and centrifuged
to separate the precipitated protein. The supernatant was injected in 2-
mL portions on a preparative RP-18 column and eluted with 20 mm HCl
in water/acetonitrile (95:5 v/v) in an isocratic manner at a 5 mLminÀ1
flow rate. Fractions of subsequent separations with the same retention
time (9–12 min for 1,2,3,5-tetrahydroxybenzene and 16–19 min for phloro-
glucinol) were combined and the products were obtained after freeze
drying.
Analytical methods: The 1H and 13C NMR spectra were recorded on a
Bruker spectrometer operating at 400 and 100 MHz, respectively. Mass
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Transhydroxylase Mechanism
FULL PAPER
solution (20%, 3”100 mL). The organic phase was dried over anhydrous
Na2SO4 and filtered, then the solvent was removed under vacuum. The
crude product was purified by column chromatography on silica gel by
using dichloromethane as the eluent; this yielded pure 3,4,5-trimethoxy-
bromobenzene (9.25 g, 75%).
nted and purged with argon. A portion (2 mL) of the mixture was puri-
fied on a preparative RP-18 column by using HCl (20 mm) in water/ace-
tonitrile (95:5 v/v) as the eluent at a 5 mLminÀ1 flow rate. Fractions with
retention times between 9–14 min for 1, 30–45 min for 6, and 20–35 min
for 7 were collected and the pure products were obtained after freeze
drying.
Synthesis of 2,4,6-trimethoxyphenol (3): A solution of 3-chloroperoxy-
benzoic acid (mCPBA, 8.6 g, 50 mmol) in dichloromethane (140 mL) was
added dropwise to a cooled (ice-bath) solution of 2,4,6-trimethoxybenzal-
dehyde (5 g, 25.5 mmol) in dry dichloromethane (30 mL) over approxi-
mately 90 min. The reaction was complete after 3 h at room temperature.
The reaction mixture was washed with saturated NaHCO3 solution (3”
25 mL), dried over anhydrous MgSO4, and concentrated under reduced
pressure. The crude product was dissolved in methanol (50 mL) and
cooled to 08C, then KOH solution (5 g in 25 mL water) was added within
20 min. The reaction was complete in 45 min at 08C. The pH value of the
solution was adjusted to 2 by addition of HCl solution (2m). The reaction
mixture was extracted with diethyl ether (3”200 mL), the etheric phase
was washed with water (3”100 mL), and the organic layer was dried
over anhydrous Na2SO4. The solvent was removed by distillation under
reduced pressure and the crude product was purified by column chroma-
tography on silica gel by using a dichloromethane:acetone mixture (95:5
v/v) as the eluent. The pure 2,4,6-trimethoxyphenol (2.34 g, 50%) was
crystallized in 2–3 d.
1,2,3,5-Tetrahydroxybenzene (1): Yield 52%; 1H NMR (D2O): d=
5.93 ppm (s, 2H); 13C NMR (D2O): d=95.5, 125.4, 145.3, 146.4 ppm;
HRMS: m/z: calcd for C6H6O4: 142.0247 [M+1]+; found: 142.0266.
2,4,6,3’,4’,5’-Hexahydroxydiphenyl ether (6): Amorphous white powder;
1
yield 58%; H NMR (D2O): d=5.92 (s, 2H), 5.98 ppm (s, 2H); 13C NMR
(D2O): d=94.2, 94.8, 123.9, 127.1, 146, 151.1, 152.1, 154.6 ppm; HRMS:
m/z: calcd for C12H11O7: 267.0505 [M+1]; found: 267.0505.
3,4,5,3’,4’,5’-Hexahydroxydiphenyl ether (7): Yield 78%; HRMS: m/z:
calcd for C12H11O7: 267.0501 [M+1]+; found: 267.0505; 1H NMR (D2O):
d=5.97 ppm (s, 4H); 13C NMR (D2O): d=97.7, 128.3, 146.1, 150.5 ppm.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft
(DFG) Priority Program “Novel Reactions and Catalytic Mechanisms in
Anaerobic Microorganisms” and the Fonds der Chemischen Industrie.
We thank P.M.H. Kroneck and B. Schink for a sample of the transhy-
droxylase.
Preparation of hexamethoxydiphenyl ethers 4 and 5: 2,4,6-Trimethoxy-
phenol (3) or 3,4,5-trimethoxyphenol (2.46 g, 13.4 mmol), 1-bromo-3,4,5-
trimethoxybenzene (2; 6.62 g, 26.8 mmol), and CuO (4.3 g, 30 mmol)
were refluxed in 2,4,6-collidine (35 mL) for 63 h under argon. The cooled
reaction mixture was treated with half-concentrated HCl (concentrated
HCl/water 1:1, 150 mL) and extracted with diethyl ether (4”200 mL).
The combined organic layers were washed with half-concentrated HCl
(500 mL), then with KOH solution (20%, 750 mL), and finally with
water. The dried organic layer was concentrated under reduced pressure
and the crude product was purified by column chromatography on silica
gel (dichloromethane/acetone (90:10 v/v)) to yield 4 or 5 (according to
the starting material).
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FEMS Microbiol. Rev. 1999, 22, 489–501.
2,4,6,3’,4’,5’-Hexamethoxydiphenyl ether (4): Amorphous white powder;
yield 41%; 1H NMR (CD3OD): d=3.75 (s, 6H), 3.78 (s, 3H), 3.79 (s,
6H), 3.84 (s, 3H), 6.12 (s, 2H), 6.23 ppm (s, 2H); 13C NMR (CD3OD):
d=55.5, 55.9, 56.3, 60.9, 91.8, 92.2, 125.9, 132.6, 153.6, 153.9, 155.3,
157.6 ppm; HRMS: m/z: calcd for C18H23O7: 351.1444 [M+1]+; found:
351.1450.
3,4,5,3’,4’,5’-Hexamethoxydiphenyl ether (5): Amorphous white powder;
yield 48%; 1H NMR (CD3OD): d=3.67 (s, 12H), 3.81 (s, 6H), 6.28 ppm
(s, 4H); 13C NMR (CD3OD): d=56.2, 61.0, 96.3, 134, 153.4, 153.9 ppm;
HRMS: m/z: calcd for C18H23O7: 351.1444 [M+1]; found: 351.1446.
[10] M. Boll,
B Schink, A. Messerschmidt, P. M. H. Kroneck, Biol.
Chem. 2005, 386, 999–1006.
[11] K. W. Glombitza, H. U. Rçsner, Phytochemistry 1974, 13, 1245–
1247.
[12] K. W. Glombitza, S. M. Li, Phytochemistry 1991, 30, 2741–2745.
[13] J. F. W. McOmie, M. L. Watts, D. E. West, Tetrahedron 1968, 24,
2289–2292.
Preparation of 1,2,3,5-tetrahydroxybenzene (1) and of the hexahydroxy-
diphenyl ethers 6 and 7: Boron tribromide solution in dichloromethane
(1m, 18.8 mL) was added to a solution of 3,4,5-trimethoxyphenol and
hexamethoxydiphenyl ether
4 or 5 (1.88 mmol) in dichloromethane
(10 mL) at À808C under argon. After being stirred for 1 h at À808C, the
reaction mixture was left to warm to room temperature. After 10 h, the
solution was cooled to 08C and water (5 mL) was added. After removal
of the dichloromethane under reduced pressure, the remaining mixture
was extracted with ethyl acetate (3”50 mL). The combined organic
phases were dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The crude product was treated with water
(20 mL) and the suspension was centrifuged. The supernatant was deca-
[14] K. W. Glombitza, U. Wçwler-Rieck, Liebigs Ann. Chem. 1988, 261–
265.
[15] D. Baas, J. RØtey, Eur. J. Biochem. 1999, 265, 896–901.
[16] A. Messerschmidt, H. Niessen, D. Abt, O. Einsle, B. Schink, P. M. H.
Kroneck, Proc. Natl. Acad. Sci. USA 2004, 101, 11571–11576.
Received: July 20, 2006
Published online: January 3, 2007
Chem. Eur. J. 2007, 13, 2805 – 2811
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2811