Structural diversity of peroxidase-catalyzed oxidation products of o-methoxyphenols

Article abstract of DOI:10.1021/ol049448l

Equation presented. The biocatalytjc oxidation of o-methoxyphenolic compounds led to a variety of oligophenols (dimers to pentamers) and some of their oxidation products. The reaction was carried out in an aqueous medium at room temperature with hydrogen peroxide as the terminal oxidant in a facile and green route to potentially bioactive compounds. Detailed structural information on the products of peroxidase-catalyzed oxidation of o-methoxyphenols is presented for the first time.

Full text of DOI:10.1021/ol049448l

ORGANIC
LETTERS
2
004
Vol. 6, No. 12
975-1978
Structural Diversity of
Peroxidase-Catalyzed Oxidation
Products of o-Methoxyphenols
1
†
†
†
†,‡
Sylvain Antoniotti, Lakshmi Santhanam, Disha Ahuja, Michael G. Hogg, and
,†,§
Jonathan S. Dordick*
Department of Chemical and Biological Engineering and Department of Biology,
Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180, and
Samuel Stratton VA Medical Center, 113 Holland AVenue, Albany, New York 12208
dordick@rpi.edu
Received March 24, 2004
ABSTRACT
The biocatalytic oxidation of o-methoxyphenolic compounds led to a variety of oligophenols (dimers to pentamers) and some of their oxidation
products. The reaction was carried out in an aqueous medium at room temperature with hydrogen peroxide as the terminal oxidant in a facile
and green route to potentially bioactive compounds. Detailed structural information on the products of peroxidase-catalyzed oxidation of
o-methoxyphenols is presented for the first time.
Peroxidases catalyze the one-electron oxidation of phenols
shown to be an inhibitor in ViVo of neutrophil and endothelial
cell NADPH oxidases. This enzyme is the primary source
1
4-6
2 2
in the presence of H O . Subsequent, mainly nonenzymatic,
radical-driven transformations result in a wide diversity of
reaction products. o-Methoxyphenols (guaiacols) are a major
class of peroxidase substrates and represent a common
structural motif found in nature, ranging from lignin precur-
of reactive oxygen species (ROS), which are implicated in
inflammatory diseases, including atherosclerosis, cancer,
diabetes, and ischemia-reperfusion lung injury.7
-11
Thus,
NADPH oxidase and its inhibition by apocynin and related
o-methoxyphenols have biomedical significance. These stud-
2
sors (e.g., coniferyl alcohol and ferulic acid ) to vanillin and
related compounds. In addition to their natural properties,
o-methoxyphenols have been shown to possess medicinal
properties. For example, a combinatorial synthetic approach
recently led to identification of a series of o-methoxyphenols
that induced apoptosis in leukemia U-937 cells. Likewise,
apocynin (4-hydroxy-3-methoxyacetophenone) has been
(
4) Holland, J. A.; O’Donnell, R. W.; Chang, M.-M.; Johnson, D. K.;
Ziegler, L. M. Endothelium 2000, 7, 109-119.
(5) Johnson, D. K.; Schillinger, K. J.; Kwait, D. M.; Hughes, C. V.;
McNamara, E. J.; Ishmael, F.; O’Donnell, R. W.; Chang, M.-M.; Hogg,
M. G.; Dordick, J. S.; Santhanam, L.; Ziegler, L. M.; Holland, J. A.
Endothelium 2002, 9, 191-203.
3
(
6) Stolk, J.; Hiltermann, T. J. N.; Dijkman, J. H.; Verhoeven, A. J. Am.
J. Respir. Cell Mol. Biol. 1994, 11, 95-102.
7) t’Hart, B. A.; Simons, J. M.; Knaan-Shanzer, S.; Bakker, N. P. M.;
(
†
Department of Chemical and Biological Engineering, Rensselaer
Labadie, R. P. Free Radical Biol. Med. 1990, 9, 127-131.
(8) Dodd, J. M.; Pearse, D. B. Am. J. Physiol. 2000, 279, H303-H312.
(9) Peters, E. A.; Hiltermann, J. T. N.; Stolk, J. Free Radical Biol. Med.
2001, 31, 1442-1447.
Polytechnic Institute.
‡
Samuel Stratton VA Medical Center.
Department of Biology, Rensselaer Polytechnic Institute.
§
(
(
(
1) Dunford, H. B. Coord. Chem. ReV. 2002, 233-234, 311-318.
2) Higuchi, T. P. Jpn. Acad. B: Phys. 2003, 79B, 227-236.
3) Nesterenko, V.; Putt, K. S.; Hergenrother, P. J. J. Am. Chem. Soc.
(10) Thabut, G.; El-Benna, J.; Samb, A.; Corda, S.; Megret, J.; Leseche,
G.; Vicaut, E.; Aubier, M.; Boczkowski, J. J. Biol. Chem. 2002, 277,
22814-22821.
2
003, 125, 14672-14673.
(11) Cotter, M. A.; Cameron, N. E. Life Sci. 2003, 73, 1813-1824.
1
0.1021/ol049448l CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/07/2004

Table 1. Products from the SBP-Catalyzed Oligomerization of ortho-Methoxyphenols
productsa
monomerb
pH
II
IIHy
IIIHy
IIIHy 2
18%
IV-2 Me
IVHy
IIIQ
III
IIIHyQ
IV
1
2
3
4
5
6
7
8
9
A
A
A
V
V
V
G
G
G
8
7
6
8
7
6
8
7
6
5%
13%
7%
1%
3%
4%
4%
4%
3%
2%
7%
40%
9%
5%
2%
18%
15%
4%
7%
2%
4%
8%
3%
11%
6%
1%
16%
12%
32%
1%
1%
2%
11%
14%
6%
5%
6%
monomerb
(continued)
pH
IVQ
IVQ-2Me
IVQ-Me
V
IVHy 2
IVQ-4M e
VQ2-4Me
VHy
VQ
VQ-Me
1
A
A
A
V
V
V
G
G
G
8
7
6
8
7
6
8
7
6
3%
9%
1%
2
3
4
5
6
7
8
9
10%
4%
4%
4%
5%
1%
27%
35%
50%
10%
6%
5%
4%
5%
6%
9%
2%
5%
5%
7%
5%
7%
9%
4%
4%
6%
5%
5%
9%
4%
8%
7%
5%
a
Proportion of main products in the first precipitate obtained after centrifugation for 30 min at 3000 rpm, determined by HPLC/UV (λ ) 254 nm).
Symbols: II, III, IV, V ) dimer, trimer, tetramer, pentamer, respectively. Q ) quinone (Q2 ) 2 quinones), Hy ) hydroxylated (Hy2 ) twice hydroxylated),
b
-
Me ) demethylated (-2Me ) twice demethylated). A ) apocynin, V ) vanillin, G ) 4-methylguaiacol. Additional precipitates give similar product
distributions.
ies have proposed that apocynin is not a direct inhibitor but
solvents16,17 or the presence of surfactants.18 Because our
interest was focused on small oligophenols (2-5 units), the
presence of 1% (v/v) DMF was deemed sufficient. The
reactions proceeded for 12 h, following which the solution
was centrifuged to recover the precipitated products. The
overall yield of apocynin oxidation products was >80% with
a very high conversion (see Supporting Information).
The precipitate was then fractionated via flash chroma-
tography over silica to yield a range of products that were
is metabolized by peroxidases in ViVo to yield oligomeric
1
2,13
oxidation products that possess biological activity.
Despite the emergence of apocynin and related compounds
as potential prodrugs against the NADPH oxidase target, the
products of peroxidase-catalyzed apocynin oxidation both
in Vitro and as metabolites of in ViVo peroxidase action
remain poorly characterized. For this reason, we embarked
on a study of the peroxidase-generated oxidation products
of apocynin and related o-methoxyphenols, paying particular
attention to oligomeric species that may hold promise as
therapeutic candidates.
Peroxidase-catalyzed oxidation of apocynin was performed
as follows, using soybean peroxidase (SBP) as a com-
mercially available and highly active enzyme:14 1.0 µg/mL
SBP was dissolved in 0.5 L of aqueous buffer (20 mM, pH
1
13
analyzed by LC-MS (APCI), UV spectroscopy, and H/ C
NMR. Positive- and negative-mode LC-MS yielded proton
and sodium adducts (M + 1 and M + 23, respectively) and
the conjugated base (M - 1) of the multiple products. As a
result, oligomers were identified to be formed via both
ortho-ortho and ortho-meta (to the phenolic moiety) coup-
ling and in some cases contained hydroxylated and quinone
products. A small fraction of demethylated products were
obtained through further peroxidase catalysis, which likely
led to the corresponding catechols or to quinone compounds.
These kinds of products are consistent with known peroxi-
6
, 7, or 8) containing 12 mM apocynin and 1% (v/v) DMF
to aid in substrate solubility). The reactions were initiated
by the slow feed (0.1 mL/h from a 30%, w/v, H solution)
of H to the reaction flask, which also prevented oxidant-
(
2 2
O
2
O
2
15
19-23
induced inactivation of SBP. The molecular weight of
peroxidase-generated oligomers and polymers is strongly
dependent on the presence and concentration of organic
dase chemistries.
The oxidation of apocynin was strongly influenced by
reaction pH. Slightly acidic conditions favored the formation
of the dimer, diapocynin, as the primary product (Table 1),
(
12) Van den Worm, E.; Beukelman, C. J.; Van den Berg, A. J. J.; Kroes,
B. H.; Labadie, R. P.; Van Dijk, H. Eur. J. Pharmacol. 2001, 433, 225-
30.
13) Simons, J. M.; t’Hart, B. A.; Ip Vai Ching, T. R.; Van Dijk, H.;
Labadie, R. P. Free Radical Biol. Med. 1990, 8, 251-258.
14) McEldoon, J. P.; Pokora, A. R.; Dordick, J. S. Enzyme Microb.
Technol. 1995, 17, 359-65.
(15) Nicell, J. A.; Wright, H. Enzyme Microb. Technol. 1997, 21, 302-
310.
(16) Dordick, J. S.; Marletta, M. A.; Klibanov, A. M. Biotechnol. Bioeng.
1987, 30, 31-6.
(17) Oguchi, T.; Tawaki, S.-i.; Uyama, H.; Kobayashi, S. Bull. Chem.
Soc. Jpn. 2000, 73, 1389-1396.
2
(
(
1976
Org. Lett., Vol. 6, No. 12, 2004

Scheme 1. Enzyme-Catalyzed Radical Formation Followed by Nonenzymatic Ortho Coupling in Solution
although several trimeric species were obtained. A small
fraction of tetramers and pentamers were also formed. The
reaction at pH 7 shifted the product spectrum to a nearly
equal fraction of trimers and tetramers, including a large total
fraction of hydroxylated products. While not unknown in
resulting from the oxidation to quinone, and two phenolic
moieties. All these functional groups are conjugated to each
other on the aromatic trimeric backbone with the five
carbonyl groups virtually locating on any of the seven C-O
bonds available.
peroxidase chemistry,20,21 hydroxylation reactions in the H
O
2 2
The C NMR spectra of compound IIIHyQ did not reveal
13
1
system are not common and may be a result of the unique
characteristics of apocynin. Products of the pH 8 reaction
were different still, and a major trimeric hydroxylated qui-
none was obtained in 40% yield (compound IIIHyQ in Table
corresponding quaternary carbons. The H NMR spectra did
not resolve the location of the specific hydroxyls oxidized
to quinones. Four signals were observed as doublets between
7.70 and 7.20 ppm accounting for four hydrogen atoms. We
took these signals as evidence of a central position for the
1). This quinone had an m/z value of 508 and the potential
4
structure shown in Scheme 2. To explain the formation of
this compound, we propose first the usual formation of the
trimer III by radical coupling. This trimer may then be
hydroxylated to give IIIHy followed by oxidation to IIIHyQ.
The lack of IIIQ at pH 8 suggests that this compound is not
likely to be along the major route to IIIHyQ.
The tautomeric equilibrium of polyphenolic quinones has
been well described.24-26 IIIHyQ contains three carbonyl
groups from the apocynin side chain, two carbonyl groups
added hydroxyl group in IIIHyQ to give a unique J coupling
for each proton. Methyl group (three methoxy and three
methyl ketone) chemical shifts were observed as expected,
1
13
in both H and C NMR analysis. The quinone products
are significant, because of their potential reactivity toward
2
7
critical thiol residues in biological targets such as critical
sequences of the protein subunits of NADPH oxidase.
In addition to reaction pH, we evaluated the influence of
the nature of the R substituent in the para position of the
Scheme 2. Secondary Reactions Likely to Occur during the Peroxidase-Catalyzed Oxidation of ortho-Methoxy Phenols (Case of a
Trimer of Apocynin, Leading to Compound IIIHyQ of Mass 508)a
a
Dashed lines on the top route indicate that it is proposed to be a minor route to IIIHyQ.
Org. Lett., Vol. 6, No. 12, 2004
1977

phenolic monomer on peroxidase-catalyzed o-methoxyphenol
oxidation. To that end, we studied the oxidation of vanillin
the side chain. An obvious difference lies in the electron-
donating/withdrawing character of the para substituent.
4-Methylguaiacol has an electron-donating substituent and
leads to relatively simple dimers, trimers, and higher oligo-
mers. Apocynin and vanillin contain electron-withdrawing
para substituents, which appear to favor quinone formation
and demethylation. We and others have performed Hammett
(4-hydroxy-3-methoxybenzaldehyde) and 4-methylguaicol.
Reactions were performed under conditions identical to those
used for apocynin at pH 6-8. As summarized in Table 1,
vanillin yielded a product spectrum that was significantly
distinct from that obtained from apocynin. At all three pHs,
vanillin underwent oxidation to predominantly quinone
products with minimal hydroxylation. Unlike apocynin,
significant ortho demethylation was observed (anywhere
from 53-72% of the converted vanillin was oxidized to a
demethylated product), such that at pH 6, ca. 50% of the
vanillin oxidation products was a didemethylated tetrameric
quinone (IVQ-2Me).
2
8,29
analysis of peroxidase catalysis.
In aqueous solutions,
the Hammett coefficient is negative, indicating increased
enzyme activity on electron-donating para substituents. Most
of these substrates undergo ortho-ortho coupling to give
simple oligomers and polymers. The less reactive electron-
withdrawing substituents also undergo oligomerization; how-
ever, their slower oxidation rate may favor noncoupling
reactions such as demethylation, hydroxylation, and oxidation
to quinones (mainly 1,4-quinones, but 1,2-quinones are
possible following demethylation).
In contrast, the major products from the oxidation of
4-methylguaiacol, which bears an electron-donating side
chain, were simple oligomers (dimer II and trimer III) with
relatively little influence of reaction pH (Figure 1).
Further work is needed to elucidate the precise mechanism
of product formation. In any event, the uniqueness of the
o-methoxy group is highlighted by using the apocynin
analogue 4-hydroxy-3-methylacetophenone. This compound,
which was poorly reactive (98% of the starting material was
recovered unchanged), formed primarily the dimer and did
3
0
not produce a complex mixture of oligomers.
In summary, peroxidase catalyzes the oxidation of o-meth-
oxyphenols to a wide array of oligomeric products, including
demethylated, quinones, and demethylated quinones. This
structural richness appears to be promoted by electron-with-
drawing substituents in the para position. The combination
of the two has served nature well as a common structural
motif for a range of biologically important compounds, in-
cluding some with therapeutic potential.
Acknowledgment. This work was supported by the
National Institutes of Health (GM66712). The authors are
grateful to Dr. Dmitri Zagorevski for his help with the mass
spectrometer (Grant CHE 0091892).
Figure 1. Final percentage of the total product of dimer, trimer,
tetramer, and pentamer as a function of the substrate (4-meth-
ylguaiacol, apocynin, vanillin).
Note Added after ASAP. One author name, Lakshmi
Santhanam, was missing in the version posted ASAP May
7
, 2004; the corrected version was posted May 13, 2004.
The different product spectra obtained from these three
closely related peroxidase substrates is due to the nature of
Supporting Information Available: Detailed general
procedure for SBP-catalyzed oxidation of phenols, full MS
data for compounds given in Table 1, and spectral data of
selected compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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