Cleavage of nonphenolic b-1 diarylpropane lignin model dimersEvidence for a hydrogen abstraction mechanism G.. Gold Department of Biochemistry and Molecular Biology, OGI School of Scienc
Trang 1Cleavage of nonphenolic b-1 diarylpropane lignin model dimers
Evidence for a hydrogen abstraction mechanism
G Vijay B Reddy1, Malayam Sridhar2and Michael H Gold
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at OHSU, Beaverton, Oregon, USA
Purified manganese peroxidase (MnP) from
Phanerocha-ete chrysosporium oxidizes nonphenolic b-1 diarylpropane
lignin model compounds in the presence of Tween 80,and
in three- to fourfold lower yield in its absence In the presence
of Tween
80,1-(3¢,4¢-diethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl)propane (I) was oxidized to
3,4-diethoxy-benzaldehyde (II),4-methoxyacetophenone (III) and
1-(3¢,4¢-diethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)pro-pane (IV),while only 3,4-diethoxybenzaldehyde (II) and
4-methoxyacetophenone (III) were detected when the
reac-tion was conducted in the absence of Tween 80 In contrast
to the oxidation of this substrate by lignin peroxidase (LiP),
oxidation of substrates by MnP did not proceed under
anaerobic conditions When the dimer (I) was deuterated at
the a position and subsequently oxidized by MnP in the
presence of Tween 80,yields of 3,4-diethoxybenzaldehyde,
4-methoxyacetophenone remained constant,while the yield
of the a-keto dimeric product (IV) decreased by
approxi-mately sixfold,suggesting the involvement of a hydrogen
abstraction mechanism MnP also oxidized the a-keto di-meric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III),in the presence and,in lower yield,in the absence of Tween 80 When the reaction was performed in the presence of 18O2,both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of18O Finally,MnP oxidized the substrate 1-(3¢,5¢-dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl) propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3¢,5¢-dimethoxyphe-nyl)-1-oxo-2-(4¢-methoxyphenyl)propane (X) In sharp contrast,LiP was not able to oxidize IX Based on these results,we propose a mechanism for the MnP-catalyzed oxidation of these dimers,involving hydrogen abstraction at
a benzylic carbon,rather than electron abstraction from an aromatic ring
Keywords: manganese peroxidase; hydrogen abstraction; diarylpropane dimers; Mn(III); radical mediator
Lignin is a complex,random,phenylpropanoid polymer
that constitutes 15–30% of woody plant cell walls [1]
White-rot basidiomycetous fungi are primarily responsible
for the initial decomposition of lignin in wood [2–5] When
cultured under ligninolytic conditions,the best-studied
white-rot basidiomycete, Phanerochaete chrysosporium,
produces two extracellular peroxidases,lignin peroxidase
(LiP) and manganese peroxidase (MnP),which,along with
an H2O2-generating system,appear to be the major
components of its lignin degradation system [2,3,6–10]
LiP oxidizes a variety of lignin model compounds,including
the most prevalent nonphenolic b-aryl ether (b-O-4 type) as
well as diarylpropane (b-1 type) structures [10–14] The enzyme abstracts one electron from the aromatic ring to form an aryl cation radical [3,11,15,16] Chemical and ESR spectroscopic evidence have confirmed the formation of cation radical species in the LiP-catalyzed oxidation of alkoxybenzenes [17–21] In contrast,MnP oxidizes Mn(II), its primary substrate,to Mn(III),which is chelated by organic acids such as oxalate or malonate [9,22,23] The Mn(III)–organic acid complex,in turn,oxidizes monomeric phenols and phenolic lignin models via formation of a phenoxy radical [9,24–27] MnP is also capable of oxidizing nonphenolic lignin model dimers and veratryl alcohol,in the presence of a radical mediator [28–30] White-rot fungi, which produce MnP and laccase but not LiP,are still able to degrade lignin efficiently [31–33],suggesting that these fungi may produce mediators,enabling MnP and/or laccase to cleave nonphenolic lignin substructures Both glutathione [28] and Tween 80 have been examined as possible mediators,and a peroxy radical has been implicated in the Tween 80 reaction [29,30]
In this study,we show that MnP oxidizes nonphenolic diarylpropane lignin models,in the presence and to a lesser extent in the absence of Tween 80 Previously,two mechanisms were considered for the oxidation and cleavage
of nonphenolic lignin dimers by MnP,electron abstraction, and hydrogen abstraction [28–30] In our current work,
we have attempted to differentiate between these two
Correspondence to M H Gold,Department of Biochemistry and
Molecular Biology,OGI School of Science and Engineering at OHSU,
20000 NW Walker Road,Beaverton,OR 97006–8921,USA.
Fax: + 1 503 7481464; Tel.: + 1 503 6460957;
E-mail: mhgold@myexcel.com
Abbreviations: LiP,lignin peroxidase; MnP,manganese peroxidase;
THF,tetrahydrofuran.
1
Present address: Merck,PO Box 2000,RY80L-109,Rahway,NJ
07065,USA.
2 Present address: Department of Chemistry & Biochemistry,Texas
Tech University,Lubbock,TX 79409,USA.
(Received 1 August 2002,revised 31 October 2002,
accepted 21 November 2002)
Trang 2mechanisms The products formed from the oxidation
and cleavage of several different diarylpropane substrates
under both aerobic and anaerobic conditions were
exam-ined In addition,we have used 18O2 to follow the
incorporation of molecular oxygen into the products Based
on the substrates used,the products identified,and the
results of stable isotope studies,we propose a mechanism
for Ca–Cb cleavage of the dimers involving hydrogen
abstraction Furthermore,our results do not support the
alternative mechanism [30],involving electron abstraction
from the aromatic ring We also show that a-keto
diaryl-propane lignin dimeric compounds are degraded by a
hydrogen abstraction mechanism to produce benzoic acid
derivatives
Materials and methods
Synthesis of substrates and products
The diarylethane model
1-(3¢,4¢-diethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)ethane was prepared as described
previ-ously [34,35] This ketone was treated with methyl iodide in
the presence of potassium tertiary butoxide and anhydrous
dimethylsulfoxide to obtain the a-keto diarylpropane model
1-(3¢,4¢-diethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)pro-pane (IV) [12] The ketone dimer IV was reduced with either
NaBH4 or NaBD4 to obtain
1-(3¢,4¢-diethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl)propane (I) or [Ca-2H1] I,
I(D),respectively Crude products were purified by silica gel
preparative thin layer chromatography,using 5% ethyl
acetate in hexanes
1-(3¢,4¢-Diethoxyphenyl)-1,3-dihydroxy-2-(4¢-methoxyphenyl)propane
(VI),1-(3¢,4¢-diethoxyphe-nyl)-1-oxo-2-(4¢-methoxyphenyl)-3-hydroxypropane (VIII)
[9,26], 1,2-dihydroxy-1-(4¢-methoxyphenyl)ethane,and
1-(4¢-methoxyphenyl)-1-oxo-2-hydroxyethane (VII) were
prepared as previously described [34,35]
1-(3¢,5¢-Dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl)ethane
This product was prepared by the Barbier reaction [36]
To a mixture of magnesium turnings (1.1 mg atom) and
3,5-dimethoxybenzaldehyde (1.1 mmol) in anhydrous
tetra-hydrofuran (THF; 10 mL) was slowly added
4-meth-oxybenzyl chloride (1 mmol; 0.14 mL) using a syringe at
room temperature under a nitrogen atmosphere and the
mixture was refluxed for 24 h The reaction was quenched
with water (10 mL) and extracted with ether (3· 5 mL)
The organic layer was washed with water (1· 10 mL),
dried over anhydrous sodium sulfate,rotary evaporated,
and the crude preparation was purified by column
chro-matography on neutral alumina,using 5% ethyl acetate in
hexanes
1-(3¢,5¢-Dimethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)ethane
The pyridinium chlorochromate-on-alumina reagent [37]
(0.75 mmol; 0.8084 g) was added to a flask containing a
solution of
1-(3,5-dimethoxyphenyl)-1-hydroxy-2-(4¢-meth-oxyphenyl)ethane (0.5 mmol) in hexanes (5 mL) After
stirring for 4 h at room temperature,the solution was
filtered,and washed with diethyl ether (3· 5 mL) The combined filtrates were evaporated to obtain the product
1-(3¢,5¢-Dimethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)propane (X) n-BuLi (0.48 mmol,0.30 mL of 1.6Msolution in hexanes) was added to a solution of diisopropylamine (0.48 mmol, 0.05 g) in dry THF (2 mL) under a nitrogen atmosphere at
0C and the mixture was stirred for 0.5 h [38] A solution of 1-(3¢,5¢-dimethoxyphenyl)-1-oxo-2-(4¢-methoxyphenyl)-ethane (0.4 mmol) in dry THF (3 mL) was added at 0C and stirred for 1 h Iodomethane (1.6 mmol) was added
at 0C,and the mixture was stirred for 8 h at room temperature The reaction was quenched with water (5 mL), extracted with ether (3· 5 mL),dried over anhydrous sodium sulfate,and concentrated by evaporation The crude preparation was purified by column chromatography on silica gel using 3% ethyl acetate in hexanes
1-(3¢,5¢-Dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxyphenyl)-1-propane (IX)
To a solution of 1-(3,5-dimethoxyphenyl)-1-oxo-2-(4-meth-oxyphenyl)propane (X) (0.2 mmol,0.06 g) in ethanol (5 mL) was added an excess of sodium borohydride (1.0 g) in three portions and the reaction mixture was stirred for 4 h at room temperature The reaction mixture was neutralized with dilute hydrochloric acid,extracted with ether (3· 5 mL),dried over anhydrous sodium sulfate and concentrated by rotary evaporation The crude preparation
of IX was purified by column chromatography on neutral alumina as described above
Chemicals 3¢,4¢-Diethoxybenzaldehyde (II),4¢-methoxyacetophenone (III),3¢,4¢-diethoxybenzoic acid (V) and 3¢,5¢-dime-thoxybenzaldehyde (XI) were obtained from Aldrich.18O2 gas (99%) was obtained from Isotec Inc (Miamisburg, OH,USA) Unless specified otherwise,other aromatic compounds were purchased from Aldrich
Enzymes Manganese peroxidase (MnP) isozyme 1 and lignin peroxi-dase (LiP) isozyme H8 were purified from the extracellular medium of acetate-buffered,agitated,aerobic cultures of
P chrysosporium OGC101 (ATCC 201542) as previously reported [39,40] Purified MnP and LiP were electrophore-tically homogeneous and had an Rzvalue of 5.0 Enzyme reactions
Reactions with MnP were conducted at 28C for 15 h in
1 mL of 50 mMsodium malonate,pH 4.5,containing the dimeric substrate (180 lM),MnSO4(0.5 mM),MnP (5 lg) and Tween 80 (polyoxyethylenesorbitan monooleate) (0.1%) LiP reactions were conducted at 28C for 5 min
in 1 mL of 20 mM succinate,pH 3.0,containing the substrate (180 lM) and enzyme (5 lg) The reactions were initiated by the addition of 100 lM HO and were
Trang 3conducted under either aerobic or anaerobic conditions.
For anaerobic experiments,reaction mixtures were
evacu-ated and flushed with argon twice to ensure removal of
oxygen H2O2 was evacuated and purged with argon
separately before addition Reaction products and
unreact-ed substrates were extractunreact-ed with ethyl acetate,driunreact-ed over
anhydrous sodium sulfate,evaporated under nitrogen,and
analyzed directly by GC or as their (acetyl or trimethylsilyl)
derivatives by GC or GC-MS,as described previously [18]
Reaction mixtures were also analyzed directly by HPLC
New substrates were also analyzed by NMR
Incorporation of18O from 18O2
Reactions were carried out in 2-mL vials fitted with rubber
septa All components of the reaction mixture,except H2O2,
were added The vials were evacuated,flushed with argon,
reevacuated,and finally equilibrated with18O2 Reactions
were initiated with the addition of a deoxygenated solution
of H2O2,and incubated for 5 min (LiP reactions) or 15 h
(MnP reactions) as described [16,27] Reaction products
were extracted and prepared for analysis as described above
Chromatography and mass spectrometry
GC-MS was performed at 70 eV on a Finnigan 4500 mass
spectrometer using a Galaxy data system and fitted with a
Hewlett Packard (HP) 5790 A gas chromatograph and a
30-m fused silica column (DB-5; J & W Scientific,Folsom,
CA,USA) The oven temperature was increased from 70 to
320C at 10 C min)1 HPLC analysis of products was
conducted with an HP Lichrospher 100 RP8 column,using
a linear gradient of 0–100% acetonitrile in 0.05%
phos-phoric acid over 10 min,with a flow rate of 1 mLÆmin)1
Products were detected at 285 nm Product yields on HPLC
were quantitated using calibration curves obtained with
standards
Results
Oxidation of diarylpropane substrates by MnP
Time courses for the oxidation of diarylpropanes I and VI
by MnP,in the presence and absence of Tween 80,are
shown in Fig 1 After 12 h of incubation,over 90% of the
added diarylpropane I was oxidized in the presence of
Tween 80,while in the absence of Tween 80,24% of the
diarylpropane I was oxidized In addition,while about 30%
of the diarylpropane VI was oxidized in the presence of
Tween 80 during the 12 h incubation,in the absence of
Tween 80,only about 5% of the VI was oxidized
The products of the oxidation reactions are shown in
Table 1 and Fig 2 The products and percent yields of the
MnP oxidation of the diarylpropane I in the presence of
Tween 80 included the a-keto diarylpropane (IV,58%),
3,4-dimethoxybenzaldehyde (II, 25%) and
4-methoxyaceto-phenone (III,21%) (Fig 2A,Table 1) A small amount
( 2%) of 3,4-diethoxybenzoic acid (V) was also detected
(not shown) The oxidation products for diarylpropane I in
the absence of Tween 80 included 3,4-diethoxybenzaldehyde
(II,20%) and 4-methoxyacetophenone (III,14%) (Table 1)
No detectable amount of a-keto diarylpropane (IV) was
produced in the absence of Tween 80 For the oxidation of the diarylpropane VI,the products included the corres-ponding a-keto diarylpropane (VIII,12%),3, 4-diethoxy-benzaldehyde (II,13%) and 4-methoxyphenyl-ketol (VII, 8%) (Fig 2C,Table 1) In the absence of Tween 80,only about 5% of the added diarylpropane VI was oxidized to yield 3,4-diethoxybenzaldehyde (II, 2.5%) and 4-methoxy-phenyl-ketol (VII,1.5%) (Table 1) Again,no detectable amount of a-keto diarylpropane VIII was formed in the absence of Tween 80 (Table 1) To further examine the mechanism of diarylpropane oxidation by the MnP system, the diarylpropane IX was prepared The products of the MnP oxidation of IX in the presence of Tween 80 included the a-keto diarylpropane (X,32%),3,5-diethoxybenzalde-hyde (XI,9.7%) and 4-methoxyacetophenone (III,11.5%) (Fig 2E,Table 1) The products of the MnP oxidation of the diarylpropane IX in the absence of Tween 80 included 3,5-dimethoxybenzaldehyde (XI, 11.2%) and 4-methoxya-cetophenone (III,14.4%) No detectable amount of the a-keto diarylpropane (X) was produced in the absence of Tween 80 (Table 1) To determine possible pathways for the oxidation of the diarylpropanes,the oxidation of the intermediate a-keto diarylpropanes,described above,also were examined Oxidation of the a-keto diarylpropane IV by MnP,in the presence of Tween 80,yielded 4-methoxyac-etophenone (III,22%) and 3,4-diethoxybenzoic acid (V,13%) (Table 1) When the oxidation of the a-keto diarylpropane (IV) was carried out in the absence of Tween 80,yields of the products 4-methoxyacetophenone (III) and 3,4-diethoxybenzoic acid (V) were decreased by approxi-mately threefold (Table 1)
In the presence of Tween 80,the a-keto diarylpropane (VIII) was oxidized to 3,4-diethoxybenzoic acid (V, 1.2%) and 4-methoxyphenyl-ketol (VII,3%) (Table 1) Less than 1% yield of these products occurred when the reaction was
Fig 1 Oxidation of the diarylpropanes I (d,s) and VI (m,n) by MnP,
in the presence (d,m) and absence (s,n) of Tween 80 Enzyme reac-tions were carried out in 50 m M malonate for 12 h,extracted with ethyl acetate,and the amount of substrate remaining was quantitated by HPLC as described in the text and in the legend to Table 1.
Trang 4conducted in the absence of Tween 80 Finally,in the
presence of Tween 80,oxidation of a-keto diarylpropane (X)
resulted in the formation of 3,5-dimethoxybenzoic acid (XII,
5.0%) and the 4-methoxyacetophenone (III,6.7%) When
the reaction was conducted in the absence of Tween 80,the
same products were formed but in lower yield (Table 1)
Effect of deuterium on the oxidation of the
diarylpropane (I)
When the a-deuterated diarylpropane I(D) was oxidized by
MnP in the presence of Tween 80,the yields of the products,
3,4-diethoxybenzaldehyde (II,28%),and
4-methoxyaceto-phenone (III,21%),were similar to those obtained with the
undeuterated substrate I,whereas the a-keto diarylpropane
product (IV,9%) was formed in a sixfold lower amount
(Table 1)
Oxidation of diarylpropane substrates by LiP
Oxidation of diarylpropane I by LiP yielded
3,4-diethoxy-benzaldehyde (II,70%) and 4-methoxyacetophenone (III,
61%) in approximately 5 min (Fig 2G) The a-keto
diarylpropane IV product was not detected in this reaction
Identical results were obtained with a-deuterated
diarylpro-pane I(D) When the diarylprodiarylpro-pane (IX) was incubated with
LiP,no products were formed and the amount of substrate
remained unchanged (Table 1) Finally,when either of the
a-keto diarylpropanes (IV or X) was incubated with LiP,no
products were observed and the amount of the substrates remained unchanged (Table 1)
Diarylpropane oxidation under either18O2or argon When the oxidation of the diarylpropane I,by MnP,was performed under18O2,0.8 atoms of18O was incorporated into the 4-methoxyacetophenone (III),whereas 3, 4-dieth-oxybenzaldehyde (II) did not contain detectable amounts of
18O (Table 2) When the oxidation of a-keto diarylpropane (IV) by MnP was carried out under 18O2, 18O was incorporated into both products Approximately 0.8 atoms
of18O were incorporated into 3,4-diethoxybenzoic acid (V) and 0.75 atoms into the 4-methoxyacetophenone (III) (Table 2) The percentage of18O incorporated was estima-ted by the ratio of the M+/(M++ 2) peaks in the mass spectrum of the products Neither the diarylpropane (I) nor the a-keto diarylpropane (IV) was oxidized by MnP when the reaction was carried out under argon,either in the presence or absence of detergent (data not shown)
Discussion
Under ligninolytic conditions, P chrysosporium secretes two extracellular peroxidases,MnP and LiP,which are mainly responsible for the initial depolymerization of lignin
in wood [2,3,22,41] While both enzymes are iron heme-containing peroxidases,their detailed reaction mechanisms differ considerably LiP abstracts an electron from the
Table 1 Products obtained from the oxidation of nonphenolic diarylpropane substrates I, I(D),VI, IX and a-keto diarylpropane substrates IV, VIII, X
by MnPaor LiPb t ¼ trace.
Tween 80 (+/–)
Products formed (mol %)
Nonphenolic diarylpropane substrates I,I(D),VI,IX
a-Keto diarylpropane substrates IV,VIII,XII by MnP a or LiP b
a MnP reactions were conducted at 28 C for 15 h in 50 m M malonate,pH 4.5,containing enzyme,substrate,MnSO 4 ,and H 2 O 2 in the presence or absence of Tween 80,and the products were analyzed by HPLC and GC-MS as described in the text Amount of products formed (mol percentage) are shown Each reaction was run in triplicate; the results are the mean values.bEnzyme reactions were conducted
at 28 C for 5 min in 20 m M succinate,pH 3.0,containing enzyme,H 2 O 2 ,and substrate,and the products were analyzed by HPLC and GC-MS as described in the text Amount of products formed (mol percentage) are shown.
Trang 5substrate aromatic ring,generating an aryl cation radical,
which decomposes further by enzymatic and nonenzymatic
processes [2,3,11–13,15,16,41,42] In contrast, MnP oxidizes
Mn(II) to Mn(III) and the latter oxidizes the aromatic
substrate [2,22,24,39]
In the absence of radical mediators,MnP mainly oxidizes
phenolic lignin substructures [9,25–27] However, in the
presence of mediators,MnP is able to oxidize nonphenolic
lignin substructures [28,29] In vitro experiments
demon-strate that MnP cleaves nonphenolic b-arylether dimers in
the presence of Tween 80,an unsaturated fatty acid
containing detergent [29,30,43] In contrast, Tween 20,
which contains a saturated fatty acid,does not act as a mediator [29] These studies suggest the involvement of lipid-derived peroxy radicals as mediators in the oxidation
of nonphenolic lignin model compounds However,the detergent-mediated mechanism of oxidation is not clearly understood It has been proposed that fatty acid-based peroxy radicals can oxidize b-aryl ether lignin model compounds by abstracting either a hydrogen from the C1 position to produce a carbon-centered radical,or an electron from the aromatic ring to produce an aryl cation radical intermediate [29,30] The C1-centered radical, resulting from a-hydrogen abstraction,has been proposed
to undergo two subsequent reactions The addition of oxygen at C1 followed by loss of HOOÆyields an uncleaved a-keto dimer,which has been proposed to undergo homo-lytic C2–O fission to expel a phenoxy radical [30] Alternatively,it has been proposed that a cation radical intermediate is formed,which subsequently undergoes
Ca–Cb cleavage,similar to LiP-catalyzed oxidations [30] However,the evidence in these studies for electron abstrac-tion by a peroxy radical is not convincing Reduced glutathione also acts as a mediator in the oxidation of veratryl alcohol and nonphenolic b-ether structures by MnP Enzymatically generated Mn(III) oxidizes the thiol to thiyl radical,which initiates substrate oxidation through hydrogen,but not electron,abstraction [28]
To determine the relative importance of a-hydrogen vs electron abstraction,we studied the mechanism of oxidation
of nonphenolic diarylpropane lignin model compounds by MnP,in the presence and absence of the unsaturated fatty acid-based detergent Tween 80 As the initial products of b-ether dimer cleavage undergo further reactions [30], elucidating the initial cleavage mechanism is difficult Therefore,we selected b-1-type lignin model substructures
as substrates,because they produce relatively stable primary products,which do not undergo extensive subsequent MnP-catalyzed oxidation
The diarylpropane I is oxidized by MnP in the presence and absence of the radical mediator Tween 80; however,the oxidation rates are approximately fourfold greater in the presence of Tween 80 than in its absence In the presence of Tween 80,the diarylpropane I undergoes a–b cleavage to produce 3,4-diethoxybenzaldehyde (II) and 4-methoxy-acetophenone (III) as well as Caoxidation to produce the corresponding a-keto diarylpropane (IV),which is the dominant product In contrast,in the absence of Tween 80, diarylpropane (I) is oxidized at a slower rate to produce only
Table 2 Incorporation of18O during the oxidation of the nonphenolic diarylpropane I and the a-keto diarylpropane IV by MnP.
Substrate Product m/z 18O incorporated (%)a
I II 196 (M++ 2) –
III 152 (M + + 2) 70
I V 212 (M++ 2) 80
III 152 (M++ 2) 75
a 18 O incorporation divided by the total oxygen incorporation
· 100 Enzyme reactions were carried out in duplicate under18O 2 in
50 m M malonate,containing enzyme,substrate,Tween 80,MnSO 4 , and H 2 O 2 for 15 h at 28 C Products were extracted and quanti-tated by GC-MS as described in the text.
Fig 2 Products identified during the oxidation of nonphenolic
diaryl-propanes (reactions A, C, and E) and a-keto diarylpropanes (reactions B,
D, and F) by homogeneous MnP and LiP (G) MnP reactions were
carried out in 50 m M malonate buffer in the presence or absence of
Tween 80 for 15 h at 28 C as described in the text LiP reactions were
carried out in 50 m M succinate at 28 C for 5 min as described in the
text Products were extracted and analyzed by HPLC and GC-MS,as
described in the text Yields of products are presented in Table 1.
Trang 6the a–b cleavage products Under these conditions,the
a-keto diarylpropane product (IV) is not
observed,suggest-ing that C1 oxidation does not occur Although only about
20% of the diarylpropane (I) is oxidized by MnP in the
absence of Tween 80,this finding is surprising,because it was
previously thought that MnP is incapable of attacking
nonphenolic lignin models in the absence of radical
media-tors [28–30] However,recent evidence suggests that metal–
oxo complexes as well as Mn(III) complexes are able to
abstract an H atom from certain aromatic compounds
[44,45]
Corresponding products are formed during the oxidation
of the diarylpropane VI,although at a lower rate The lower
reactivity of VI,when compared to I,may be due to the
stearic hindrance offered by the hydroxyl group at C3,
which may inhibit hydrogen abstraction at C2 When the
MnP reaction was conducted under anaerobic conditions,
in the presence or absence of the detergent,oxidation of
these diarylpropanes does not occur,indicating that
molecular oxygen is required
The LiP oxidation of diarylpropane substructures has
been well studied and proceeds through an aryl cation
radical intermediate [11,15,16] Therefore, we compared the
LiP and MnP oxidation of the diarylpropane I under a
variety of conditions LiP oxidizes over 70% of the
diarylpropane I in 5 min to produce the Ca–Cb cleavage
products 3,4-diethoxybenzaldehyde (II) and
4-methoxy-acetophenone (III) Furthermore,unlike the MnP reaction,
the LiP-catalyzed oxidation of the diarylpropane I proceeds
efficiently under both aerobic and anaerobic conditions [16]
Finally,the a-keto diarylpropane is not formed in the
LiP-catalyzed oxidation of the diarylpropane I [12,16], whereas
it is the major product in the MnP reaction As the LiP
oxidation of the diarylpropane I proceeds by the formation
of an aryl cation radical [16],these results suggest that a
cation radical is not an intermediate in the MnP-catalyzed
reactions reported here
To pursue this question further,we examined the
oxida-tion of
1-(3¢,5¢-dimethoxyphenyl)-1-hydroxy-2-(4¢-methoxy-phenyl)-1-propane (IX) by MnP and LiP As expected,LiP
was not able to oxidize the diarylpropane IX,owing to the
lack of an electron-donating methoxy group at the para
position of the A ring This strongly suggests that a cation
radical is difficult to produce with this substrate In contrast,
in the presence or absence of Tween 80,MnP oxidizes the
diarylpropane (IX)-producing products corresponding to
those produced during the oxidation of the diarylpropane
(I) These results support our view that electron abstraction
is an unlikely mechanism for the MnP-catalyzed oxidation
of these diarylpropane substrates
The presence of deuterium at the C1 position of the
diarylpropane (I) (Fig 2A) had a strong influence on the
yield of the a-keto diarylpropane (IV) formed When
deuterated diarylpropane I(D) was used as the substrate,the
yield of the a-keto diarylpropane (IV) was decreased by
about 6 fold,suggesting the involvement of H-abstraction
However,yields of the Ca–Cb cleavage products
3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone
(III) remained almost identical,for the deuterated and
undeuterated substrates This result is in contrast to that
with the LiP-catalyzed oxidation of I(D),which is not
affected by the presence of deuterium
Oxidation of the diarylpropane I to 3,4-diethoxybenzal-dehyde (II),4-methoxyacetophenone (III),and a-keto diarylpropane (IV) can be explained on the basis of an initial hydrogen abstraction reaction at one of two posi-tions A benzylic radical can be generated at C1,which is resonance stabilized by aromatic ring A,or at C2,which is stabilized by ring B,as well as by the adjacent methyl group through hyperconjugation However,the presence of a hydroxyl group at C1 renders the C1–H bond more labile than the C2–H bond A radical generated at C1 would add
to O2to form a peroxyl radical,which would then eliminate HOOÆ to yield the a-keto diarylpropane IV,as shown in Fig 3 This mechanism is similar to that reported earlier for the thiyl radical-mediated oxidation of nonphenolic lignin dimers by MnP [28] Alternatively,a radical generated at C2 would add to O2 and the resulting peroxy radical could abstract a hydrogen atom to form an unstable hydroper-oxide The latter would undergo Ca–Cbcleavage with the elimination of H2O to form 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) (Fig 3) Addition of deuterium at the C1 position would slow the hydrogen abstraction at C1,but would have no effect on the formation of a radical at C2 as we observed
Fig 3 Proposed hydrogen abstraction mechanism for the oxidative cleavage of the diarylpropane (I) by MnP in the presence of Tween 80.
Trang 7When the oxidation of the diarylpropane I is performed
in the presence of18O2,18O is incorporated only into the
acetophenone (III) The benzaldehyde (II) did not contain
any 18O This is similar to the LiP-catalyzed oxidation
(electron abstraction),where18O is incorporated only into
benzylic radical-derived products [16] Therefore,hydrogen
abstraction at C2 and electron abstraction from the
aromatic ring result in Ca–Cbcleavage and 18O
incorpor-ation only into the acetophenone (III) The crucial
differ-ence is that the hydrogen abstraction reaction catalyzed by
MnP,unlike the LiP-catalyzed oxidation,does not proceed
in the absence of O2[16]
The a-keto diarylpropane (IV) also is oxidized by MnP in
the presence of Tween 80 The reaction products included
4-methoxyacetophenone (III) and 3,4-diethoxybenzoic acid
(V) The benzaldehyde (II) is not detected as a product of
the reaction When the reaction is carried out in the absence
of Tween 80,the oxidation rate is about 3-fold lower,but
again,4-methoxyacetophenone (III) and
3,4-diethoxyben-zoic acid (V) were detected The a-keto dimer (IV) was not
oxidized under anaerobic conditions,indicating that oxygen
is required for this reaction as well
In contrast to the results with MnP,LiP is not able to
oxidize the dimer (IX),nor any of the a-keto diarylpropane
dimers Furthermore,LiP oxidation of the diarylpropane (I)
does not yield an a-keto diarylpropane product Finally,the
LiP oxidation of diarylpropanes occurs in the absence of
molecular oxygen Because LiP oxidizes these substrates by
electron abstraction to form an aryl cation radical,these
results strongly suggest electron abstraction is not occurring
in the MnP reactions
The mechanism we propose for the MnP oxidation of
the a-keto diarylpropane (IV) is shown in Fig 4 The
C2-centered radical generated after C2–H abstraction
would add to oxygen to form a peroxy radical The latter
can react with the carbonyl group,producing an unstable
dioxetane,which decomposes to 4-methoxyacetophenone
(III) and 3,4-diethoxybenzoic acid (V) (Fig 4)
When the oxidation of a-keto diarylpropane (IV) by
MnP is conducted in the presence of18O2,18O is
incorpor-ated into 4-methoxyacetophenone (III) and
3,4-diethoxy-benzoic acid (V),supporting the mechanism shown in
Fig 4 MnP slowly oxidizes the a-keto diarylpropane (IV)
even in the absence of Tween 80 Formation of aromatic
acids from nonphenolic lignin model compounds has not
been reported previously In this study we also identified a
small amount of 3,4-diethoxybenzoic acid during the
oxidation of diarylpropane I by MnP in the presence of
Tween 80 This is most likely due to the further oxidation of
either 3,4-diethoxybenzaldehyde (II) or the
a-ketodiaryl-propane (IV) produced during the oxidation of I We have
confirmed this secondary oxidation process in a separate
reaction using 3,4-diethoxybenzaldehyde (II) as a substrate
(data not shown) The oxidation of a benzaldehyde to
benzoic acid by MnP has been discussed recently [29]
According to the mechanisms shown in Fig 4,products
formed from the Ca–Cbcleavage should be in equal molar
ratio However,we observe that yields of the acetophenone
(III) are slightly lower than for the benzaldehyde (II) It is
likely that some of the products are degraded further by the
enzyme We are investigating the nature of the oxidant in
the reaction of the diarylpropane I by MnP in the absence of
unsaturated detergent (Tween 80) However,both metal oxo complexes and Mn(III) complexes are capable of abstracting a hydrogen atom from organic compounds [44,45] In addition, a malonic acid-generated free radical may be involved in the process [46]
In conclusion,the present study shows that MnP can catalyze Caoxidation and Ca–Cbcleavage of nonphenolic diarylpropane model compounds in the presence of Tween
80 and at a lower rate in its absence Based on the products formed under various conditions,a mechanism based on electron abstraction can be ruled out; rather,these compounds apparently are oxidized solely via hydrogen abstraction mechanisms This study also shows that the a-keto-1,2-diarylpropane is oxidized via hydrogen abstrac-tion to produce an aromatic acid and an acetophenone product
Acknowledgments
This research was supported by Grants MCB-9808430 from the National Science Foundation and DE-FG03–96ER30325 from the Division of Energy Biosciences,U.S Department of Energy (to M.H.G).
Fig 4 Proposed hydrogen abstraction mechanism for the oxidative cleavage of the a-keto diarylpropane (IV) by MnP in the presence of Tween 80.
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