AbstractUnder aerobic conditions homogeneous lignin peroxidase catalyzed the oxidation of 1-(4'-methoxyphenyl)-2-(2″,5′-dimethoxy-4″-phenylphenoxy)-1,3-dihydroxypropane (I) to yield four products: 1-(4'-methoxy-phenyl)-1,2,3-trihydroxypropane (X), 4-[α-hydroxy-α-(4'-methoxyphenyl)-methyl]-1,3-dioxolane-2-one (V), 4-(4'-methoxyphenyl)-5-hydroxymethyl-1,3-dioxolane-2-one (VI) and 5-hydroxy-5-carbomethoxy-4-phenyl-oxol-3-en-2-one (VIII). V, VI and VIII are all products of ring opening reactions. When the reaction was conducted under anaerobic conditions, the substrate was oxidized but no ring-cleaved products were detected. During the oxidation of I, 4 atoms of 18O from 18O2 were incorporated into the lactol product VIII

Gold, Michael H.

Mayfield, Mary B.

Miki, Keiji

Renganathan, V.

Elsevier - Publisher Connector 

Volume 210, number 2, 199-203 FEB 04346 January 1987 
Aromatic ring cleavage of a /?-biphenyl ether dimer 
catalyzed by lignin peroxidase of ~h~~e~och~e~e 
chrysosporium 
Keiji Miki*, V. Renganathan +, Mary B. Mayfield and Michael H. Gold 
Department of Chemical and 3iofogical Sciences, Oregon Graduate Center, Beaverton, OR 97006-1999, USA 
Received 29 October 1986 
Under aerobic onditions homogeneous lignin peroxidase catalyzed the oxidation of I-(4’-methoxyphenyl)- 
2-(2”,5”-dimethoxy-4”-phenylphenoxy)-1,3-dihydroxypropane (I) to yield four products: I-(4’-methoxy- 
phenyl)-1,2,3-trihydroxypropane (X), 4-[a-hydroxy-a-(4’-methoxyphenyl)-methyl]-1,3-dioxolane-2-one (V), 
4-(4’-methoxyphenyl)-5-hydroxymethyl-l,Zdioxolane-2-one (VI) and S-hydroxy-S-carbomethoxy-4- 
phenyl-oxol-3-en-2-one (VIII). V, VI and VIII are all products of ring opening reactions. When the reaction 
was conducted under anaerobic conditions, the substrate was oxidized but no ring-cleaved products were 
detected. During the oxidation of I, 4 atoms of lSg from ‘Sg, were incorporated into the lactol product 
VIII. 
Aromatic ring cleavage; Lignin degradation; j3-Aryl ether; Lignin peroxidase; Aryl cation radical 
1. INTRODUCTION 
Recently, lignin peroxidase (Lip), a heme- 
containing, HzOz-requiring enzyme, has been 
purified from the extracellular medium of the 
fungus ~~~neroc~uete c~rysos~ori~m [l-3]. The 
HzOz-oxidized states of LIP [3,4] are similar to 
those of HRP [5]. The homogeneous enzyme ox- 
idizes a variety of lignin model compounds [l-3] 
including fl-aryl ether monomers and dimers 16-81. 
Earlier studies demonstrated that cultures of P. 
Correspondence address: M.H. Gold, Department of 
Chemical and Biological Sciences, Oregon Graduate 
Center, Beaverton, OR 97006-1999, USA 
* Present address: National Research Institute for 
Pollution and Resources, Yatabe, Tsukuba, Ibaraki 
305, Japan 
+ Present address: Smith Kline & French Laboratories, 
709 Swedeland Road, Swedeland, PA 19479, USA 
Abbreviations: Lip, lignin peroxidase; TMS-ether, tri- 
methylsilyl ether; MS, mass spectrum 
c~rysosporium oxidized lignin [9] and fl-aryl ether 
dimeric model compounds [lo] via pathways in- 
volving ring opening reactions. In this report, we 
show that the ,&aryl ether dimer l-(4’-methoxy- 
phenyl)-2-(2”,5’‘-dimethoxy-4”-phenylphenoxy)- 
1,3_dihydroxypropane (I) is oxidized by homoge- 
neous LiP to several products derived from ring 
opening reactions. 
2. MATERIALS AND METHODS 
LiP II was purified to homogeneity from ace- 
tate-buffered agitated cultures of P. c~rysos~o- 
rium [1,3]. Model compound oxidations were 
carried out at 37°C in 1 ml Na-succinate (pH 4.5) 
containing substrate (0.02%) and enzyme (5 pg). 
Reaction mixtures were purged with 02 or eva- 
cuated and flushed with argon as indicated. Reac- 
tions performed under “02 were carried out as 
described [ 111. Reactions were initiated by addi- 
tion of Hz02 (100 PM) and run for 15 min. NaCl 
was then added to saturation and the reaction mix- 
tures extracted, dried and silylated (BSTFA/py- 
Published by Elsevier Science Pubfiskers 3. V. ~B~o~edi~at Division) 
00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 199 
Volume 210, number 2 FEBS LETTERS 
ridine, 2: 1, v/v) [1,7]. Capillary GCMS was 
performed on a VG analytical 707OE instrument. 
2.1 Preparation of compounds 
pane-l-one (VII). VII and N,N’ -carbonyldiimida- 
zole in benzene, reflux; reduction with NaBH4 to 
yield a mixture of V and VI which were separated 
on silica gel (hexane, ethyl acetate). V: MS (m/z) 
(mono TMS ether) 296 (M+, 0.3%), 237 (3.5%), 
209 (lOO%), 135 (17070), 121 (7.3’70), 101 (7.4%). 
VI: MS (m/z) (mono TMS ether) 296 (M+, 3.1%), 
206 (9.2%), 162 (64.6%), 135 (85%), 121 (loo%), 
103 (17.7%). 
January 1987 
l-(4’-Methoxyphenoxy)-2-(2”,5’‘-dimethoxy-4”- 
phenylphenoxy)- 1,3_dihydroxypropane (I): 4-Phe- 
nylphenol and bromine dioxane complex in ether 
at 0°C to yield 2,6-dibromo-4-phenylphenol (II). 
MS (m/z) (mono TMS ether) 402 (M+, 10%); II 
and NaOMe (28% MeOH solution), CuI 
(catalytic) in DMF, at 120°C for 16 h yields 
2,6-dimethoxy-4-phenylphenol (III). MS (m/z) 
(mono TMS ether) 302 (M+, 42%); condensation 
of III with l-(4’-methoxyphenyl)-2-bromo-l-oxo- 
ethane, KzCOj, in acetone yields l-(4’-methoxy- 
phenyl)-2-(2’ ,6’-dimethoxy-4-phenylphenoxy) etha- 
none (IV); hydroxymethylation with HCHO 
followed by reduction with NaBH4 [12] to yield I. 
MS (m/z) (di TMS ether) 554 (M+, 0.4%), 302 
(29.8%), 230 (25.9%), 222 (23.3%), 162 (31.3%), 
133 (43.3%). 
4-[cu-Hydroxy-cu-(4’-methoxyphenyl)-methyl]-1, 
3-dioxolane-2-one (V) and 4-(4’-methoxyphenyl) 
-5-hydroxymethyl-1,3-dioxolane-2-one (VI) were 
synthesized by modification of a reported pro- 
cedure [lo]. p-Methoxyacetophenone was bro- 
minated. The product was treated with K-formate, 
reflux, for 20 h; hydroxymethylation with HCHO 
to yield l-(4’-methoxyphenyl-2,3_dihydroxypro- 
OMe 
Hos:& - 
MeOv 
VIII: 3-Phenyl-2-pentendioic acid dimethyl ester 
(IX) was prepared by a described procedure 
[13,14]. IX MS (m/z) 234 (M+, 10.9%), 202 
(54.3%), 174 (loo%), 159 (32.3%), 115 (93.4%). 
IX and one equivalent Se02 in acetic acid were stir- 
red at 120°C for 16 h to yield the lactol VIII. ‘H 
NMR G(CDC13): 3.82 (3H, s, OMe), 6.53 (lH, s, 
=CH), 7.3-7.6 (5H, m, Ar-H), 5.3 (lH, -OH). 
MS (m/z) (mono TMS ether) 306 (M+, 2.7%), 291 
(14.5%), 247 (78.7%), 219 (8.2%), 102 (39.4%), 
89 (39.9%), 73 (100%). 
X: l-(4’-Methoxyphenyl)-1,2,3-trihydroxypro- 
pane was prepared from 4-methoxycinnamate by a 
procedure described in [ 151. 
3. RESULTS 
As shown in fig. 1, under oxygen I is oxidized by 
LiP to produce four identifiable products: X (5.34 
mol%), MS (m/z) (414 M+); the lactol VIII 
HOH2C 
YO” 
f+ n OH 
+ 
Meow Meow 
I x (5.34%) VCI 0%) 
Me0 
MeOOC 
0 
,c=o 
Ho H 
x 
0 
Yl (2 12%) m (3.81%) 
Fig. 1. Oxidative cleavage of the ,&aryl ether dimer I by homogeneous lignin peroxidase. 9’0 = (mol of product form- 
ed/mol of initial substrate) x 100. 
200 
Volume 210, number 2 FEBS LETTERS January 1987 
A. 
-I+ 
I. 
IOO- 
100 x H2C’O 
’ >O 
-73 HC-0 
Cd 
I 
HC-OTMS 
50- 
2. I I I 1 
C 00 100 120 140 160 180 200 220 240 260 275 280 285 290 295 300 
z 
e 
m/z 
C 
m l? 
.1 CH2 B. 1+ 
0 :H CH2 -It CH2OTMS 
xi 
a IOO- 121 l=o E-O 
4x 
H;-o’=’ 
HC-0 
-73 135 
_ zH20TMS 
50- \ 
103 OMe 
192 
M+296 
0 I ’ I ’ I 0 ,I I 1, I I I I 
80 100 I20 140 160 180 200 220 240 260275 
I 
280 285 290 295 300 
m/t 
Fig.2. (A) Mass spectra of the TMSi derivatized V formed under 1602. (B) Mass spectra of the TMSi derivatized VI 
formed under 1602. 
69 
102 
s. 121 
.Z 
I? I I I I 
al 60 60 100 120 I40 160 190 200 220 240 260275 260 255 290 
z 
295 300 305 310 315 320 
m/z 
aa 
.1 ‘;i IOO- 73 
f 
102 
I21 
,-I. h! :‘I, 11, ,’ , , :I , , ;’ , , , , , , I I 
60 50 100 120 140 160 I60 200 220 240 260275 290 285 290 295 300 305 310 315 320 
m/z 
Fig.3. Mass spectra of the TMSi derivatized lactol VIII. (A) Reaction conducted under 1602. (B) Reaction conducted 
under ‘*Oz. 
201 
Volume 210, number 2 FEBS LETTERS January 1987 
(3.81%), the dioxolane-Zone V (l.O%), and the 
dioxolane-2-one VI (2.12%). No products were ob- 
tained when the reactions were conducted in the 
absence of Hz02 or enzyme. Ring-opened products 
were not obtained when the reaction was con- 
ducted in the absence of oxygen. 
Fig.2A shows the mass fragmentation pattern of 
the dioxolane-Zone V. An identical pattern was 
obtained when the reaction was conducted under 
“02, indicating the igO was not incorporated into 
the product. Fig.2B shows the mass fragmentation 
pattern of the dioxolane 2-one VI. When the reac- 
tion was conducted under i802, increased ions at 
298 and 208 (not shown) indicated 30 atom% in- 
corporation of the oxygen in the keto position. 
Fig.3 shows the mass fragmentation patterns of 
the lactol VIII obtained from the oxidation of I 
under I602 and under 1802. The MS of the lactol 
obtained under 1602 has a molecular ion at 306 and 
diagnostic ions at 291, 247, and 89. The MS of the 
lactol obtained under 1802 has a molecular ion at 
314 and diagnostic ions at 299, 253 and 89, in- 
dicating that a maximum of 4 atoms of oxygen 
were incorporated into the product. 
4. DISCUSSION 
Earlier studies have shown that under aerobic 
conditions, cultures of P. chrysosporium oxidize 
both lignin [9] and&aryl ether dimeric compounds 
[lo] via pathways involving ring-opening reac- 
tions. Recently, it has been shown that lignin 
peroxidase can open the aromatic ring of veratryl 
alcohol [16]. Although it has just been reported 
that this enzyme also opens rings in &aryl ether 
dimers [17,18], a mechanism has not been sug- 
gested. In this study we use a unique fi-aryl ether 
substrate containing a biphenyl derivative of ring B 
which allowed the isolation, for the first time, of 
a B-ring opened aromatic product. Dioxygenases 
are the only oxygen-mediated enzymes previously 
known to open aromatic rings. These non-heme 
iron proteins, which require o-diphenolic sub- 
strates, activate molecular oxygen during the reac- 
tion [19]. In contrast, LiP is a peroxidase [4,11, 
20,211 and is not known to activate molecular 02 
[11,20]. The results reported here, together with 
other currently available evidence on the mecha- 
nism of Lip, enable us to propose a mechanism for 
aromatic ring cleavage by this enzyme. This 
202 
mechanism involves an initial one-electron oxida- 
tion of ring B to form an aryl cation radical 
[I 1,20,21]. This intermediate undergoes one of the 
following reactions: (a) nucleophilic attack by 
either the a- or r-hydroxyl on the ether carbon of 
ring B and loss of a proton to form a 1,3-di- 
oxolane-cyclohexadiene radical structure as de- 
scribed [6,7]; (b) nucleophilic substitution by the 
a-hydroxyl on a methoxyl-bearing carbon of ring 
B with the elimination of methanol and formation 
of a benzodioxane radical intermediate [7]. The 
structural resemblance of the ring-cleaved 
products V and VI to the 1,3-dioxolane- 
cyclohexadiene radical suggests that this radical is 
an intermediate in the formation of the 
dioxolane-Zones V and VI. Earlier reports have 
described non-enzymatic ring cleavage of 
cyclohexadiene radicals in the presence of 02 [22]. 
By analogy the cyclohexadienyl radical produced 
by LiP could lead to a dioxygenase-like ring fis- 
sion, producing the 1,3-dioxolane-2-ones. This is 
supported by our observation that ring cleavage 
occurs only in the presence of 02. The lactol VIII 
is a novel product because it retains almost all of 
the carbons of ring B. During its formation, a 
maximum of 4 atoms of oxygen are incorporated 
into the product VIII. The loss of one methoxyl 
group during the formation of VIII indicates that 
this product arises from a demethoxylated in- 
termediate such as the benzodioxane described 
above. It is therefore possible that the lactol VIII 
may not be the counterpart product of V and VI. 
In contrast to dioxygenase reactions the novel ring 
cleavage proposed here appears to occur through 
the non-enzymatic reaction between an enzyme- 
produced radical and molecular oxygen. 
ACKNOWLEDGEMENTS 
Supported by grant DE-FG06-86ER13550 from 
the USDOE and by grant DMB 8311441 from the 
NSF. 
REFERENCES 
[l] Gold, M.H., Kuwahara, M., Chiu, A.A. and 
Glenn, J.K. (1984) Arch. Biochem. Biophys. 234, 
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[2] Tien, M. and Kirk, T.K. (1984) Proc. Natl. Acad. 
Sci. USA 81, 2280-2284. 
Volume 210, number 2 FEBS LETTERS January 1987 
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203 


Aromatic ring cleavage of a β-biphenyl ether dimer catalyzed by lignin peroxidase of phanerochaete chrysosporium 

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Aromatic ring cleavage of a β-biphenyl ether dimer catalyzed by lignin peroxidase of phanerochaete chrysosporium

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