Microbial transformations of p-Coumaric acid by Bacillus megaterium and Curvularia lunata

Article abstract of DOI:10.1021/np010238g

p-Coumaric acid (1) is an abundant phenolic natural product that exhibits both chemoprotectant and antioxidant properties. Microbial transformation screening studies showed that 1 was converted to 4-vinylphenol (4), 4-hydroxybenzoic acid (2), caffeic acid (5), protocatechuic acid (6), and other unidentified metabolites over 144 h. 4-Vinylphenol (4) and its dimer, (2R,2S)-4-(2,3-dihydro-5-hyroxy-2-benzofuranyl) phenol (11), were produced by Bacillus megaterium, and 5-[(E)-2-carboxyethenyl]-2,3-dihydro-2-(4hydroxyphenyl)-3- benzofurancarboxylic acid (15) and 4-hydroxybenzoic acid (2) were produced by Curvularia lunata. On the basis of deuterium-labeling experiments, B. megaterium catalyzes the nonoxidative enzymatic tautomerization of 1 to a vinylogous β-keto acid intermediate that decarboxylates to 4. The presence of peroxidase and laccase activities in C. lunata extracts suggests that these enzymes may be involved in one-electron, p-coumaric acid dimerization in this organism.

Full text of DOI:10.1021/np010238g

1
408
J . Nat. Prod. 2001, 64, 1408-1414
Micr obia l Tr a n sfor m a tion s of p-Cou m a r ic Acid by Ba cillu s m ega ter iu m a n d
Cu r vu la r ia lu n a ta
J anelle L. Torres y Torres and J ohn P. N. Rosazza*
Division of Medicinal and Natural Products Chemistry, and Center for Biocatalysis and Bioprocessing, College of Pharmacy,
University of Iowa, Iowa City, Iowa 52242
Received May 11, 2001
p-Coumaric acid (1) is an abundant phenolic natural product that exhibits both chemoprotectant and
antioxidant properties. Microbial transformation screening studies showed that 1 was converted to
4
-vinylphenol (4), 4-hydroxybenzoic acid (2), caffeic acid (5), protocatechuic acid (6), and other unidentified
metabolites over 144 h. 4-Vinylphenol (4) and its dimer, (2R,2S)-4-(2,3-dihydro-5-hyroxy-2-benzofuranyl)
phenol (11), were produced by Bacillus megaterium, and 5-[(E)-2-carboxyethenyl]-2,3-dihydro-2-(4-
hydroxyphenyl)-3-benzofurancarboxylic acid (15) and 4-hydroxybenzoic acid (2) were produced by
Curvularia lunata. On the basis of deuterium-labeling experiments, B. megaterium catalyzes the
nonoxidative enzymatic tautomerization of 1 to a vinylogous â-keto acid intermediate that decarboxylates
to 4. The presence of peroxidase and laccase activities in C. lunata extracts suggests that these enzymes
may be involved in one-electron, p-coumaric acid dimerization in this organism.
p-Coumaric acid (1) is the central intermediate in the
biosynthesis of many plant phenols, and it is present in
esterified or free acid forms in many fruits, vegetables, and
Graminaceous plants.1 Results from our laboratory indi-
cate that it composes approximately 4% of the dry weight
Ta ble 1. Results of Screening Microorganisms for Conversions
of p-Coumaric Acid (1)
straina
Ub
species
2
4
5/6
,2
Aspergillus alliaceus
A. giganteus
A. niger
Bacillus lichenformis
B. megaterium
B. pumilus
UI-315
UI-10
UI-X-172
UI-A-5A
ATCC 14581
DRVUI-DRV
ATCC 6633
IFO-3108
ATCC 16373
UI-MR-Can
NRRL 2178
ATCC 11011
NRRL 1086
UI-MR-188
NRRL 5646
ATCC 36740
UI-MR-193
ATCC 9777
ATCC 20129
ATCC 11796
UI-L-103
+
+
3
of maize plant parts. The large quantities of 1 that are
+
+
+
+
+
+
+
+
+
+
+
+
+
available naturally render it useful as a precursor for the
biocatalytic production of value-added aromatic natural
products.
B. subtilis
Although no systematic screening of microorganisms for
the purpose of identifying metabolic products of 1 has been
reported, 4-hydroxybenzoic acid (2) and protocatechuic acid
B. subtilis var. niger
Caldariomyces fumago
Candida lipolytica
Curvularia lunata
Cylindrocarpon radicicola
Gliocladium deliquescens
Memnoniella echinulata
Nocardia sp.
Penicillium notatum
P. purpurogenum
P. purpurogenum
Rhodotorula rubra
Streptomyces griseolus
S. griseus
+
+
+
+
+
4
-6
7-11
12,13
(6),
4-vinylphenol (4),
caffeic acid (5),
and 4-eth-
9
+
+
+
ylphenol have all been identified as metabolites of 1.
p-Coumaric acid hydroxylases1
2,13
7,8,14,15
+
and decarboxylases
+
+
are involved in the conversions of 1 to 5 and 4, respectively.
p-Coumaric acid was examined as a sole carbon source for
the growth of fungi and yeasts,16 but no degradation
pathway intermediates were identified.
This paper presents the first comprehensive results of
screening bacteria, yeasts, and filamentous fungi for their
abilities to transform 1 to a range of products. The
mechanism of 4-vinylphenol formation by Bacillus mega-
terium was examined, and preparative scale biotransfor-
mation reactions were used to isolate and characterize two
new phenolic dimers produced by B. megaterium and
Curvularia lunata.
+
+
+
+
+
+
+
+
a
ATCC, American Type Culture Collection, Rockville, MD;
NRRL, Northern Regional Research Laboratories, Peoria, IL; IFO,
Institute for Fermentation, Osaka, J apan; UI, University of Iowa,
College of Pharmacy, Iowa City, IA. b Unknown products by TLC.
rubra completely metabolized 1 to 4 in addition to other
products. Catechol metabolites (5 or 6) had the same TLC
mobilities and were indistinguishable from each other.
Catechols were formed by A. giganteus, C. radicicola,
Gliocladium deliquescens, and Streptomyces griseus. Caffeic
acid (5) was isolated and spectrally identified as a metabo-
lite from S. griseus (24 h, 5% yield, 16% by HPLC) and G.
deliquescens (48 h, 6% yield), which also gave 6 (48 h
isolated yield, 2%). Most organisms screened gave at least
one unknown metabolite from 1 with a range of TLC
mobilities and colors when developed plates were sprayed
with diazotized sulfanilic acid reagent.
Resu lts a n d Discu ssion
Scr een in g Stu d ies. Microbial transformation screening
studies identified cultures that reproducibly metabolized
1
to new products. On the basis of TLC analysis (system
A), 21 microorganisms (Table 1) converted 1 to 2, 4, 5, 6,
and other unidentified metabolites (Figure 1). These in-
clude oxidative decarboxylation of 1 to 2 by the fungi
Curvularia lunata, Memnoniella echinulata, and Aspergil-
lus alliaceus (each within 6 h). 4-Vinylphenol (4) was the
most commonly observed metabolite of 1. Five Bacilli,
Caldariomyces fumago, Candida lipolytica, Cylindrocarpon
radicicola, Penicillium purpurogenum, and Rhodotorula
Isola tion a n d Ch a r a cter iza tion of 4-Vin ylp h en ol.
Initial screening results were confirmed and showed that
1
was transformed by B. megaterium to 4 as the sole
metabolite within 6 h, and then at 24 h, three new products
were observed at R 0.55 (brown), R 0.45 (orange), and at
*
Corresponding author. Tel: (319) 335-4902. Fax: (319) 335-4901.
E-mail: john-rosazza@uiowa.edu.
f
f
1
0.1021/np010238g CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/26/2001

Microbial Transformations of p-Coumaric Acid
J ournal of Natural Products, 2001, Vol. 64, No. 11 1409
F igu r e 1. Compounds formed by whole cell biotransformations of p-coumaric acid (1) in the screening study and by B. megaterium cultures
incubated with p-coumaric acid in solutions containing D O.
2
Ta ble 2. GC/MS and 1H NMR Results of 4 Isolated from
Cultures of B. megaterium Incubated with 1 in H2O, H2O-D2O,
and D2O
MS % relative intensity
m/z
1H NMR relative intensity
D2O
(v/v %)
120
121
122
for Hb-2′ (δ 5.03)
0
0
00
100
100
47
5.4
39
100
0.2
3.1
8.7
1.00
0.71
0.24
5
1
R
f
0.38 (orange). When 4-vinylphenol (4) was incubated in
0.45 and
f
0.38 were observed as nonenzymatically formed arti-
uninoculated medium as a control, products at R
R
f
facts. 4-Vinylphenol (4) was isolated from preparative-scale
cultures 24 h after substrate addition. Cells were discarded
when TLC analysis of acetone extracts showed only traces
of substrate or product. The supernatant afforded a brown
oil that gave 4 as an off-white powder (13% yield) after
silica gel column chromatography. The product was chro-
1
matographically (TLC system A) and spectrally ( H NMR)
identical to standard 4.
F igu r e 2. 1H NMR spectra of selected protons of 4-vinylphenol (4)
isolated from B. megaterium decarboxylation reactions conducted in
Con ver sion of 1 to 4 a n d 4a in D
mechanism of 1 to 4 by B. megaterium was examined in
incubations containing H O, H O (1:1, v/v), or D O.
2
O. The conversion
0.1 M KH
C) D O.
2 4 2 2 2
PO prepared with (A) H O, (B) H O-D O (1:1, v/v), and
(
2
2
2
O-D
2
2
MS and NMR analyses (Table 2) of unlabeled 4 show m/z
Ta ble 3. NMR Data (methanol-d4) for
2R,2S)-4-(2,3-Dihydro-5-hydroxy-2-benzofuranyl)phenol (11)
+
1
1
20 for the molecular ion C
8
H
8
O ([M] ), and 4a shows m/z
(
+
21 for C
8
H
7
DO ([M] ). GC/MS results (Table 2) show 34%
position
1′
1H (δ, J in Hz)
13C (δ) HMBC correlations (C#)
and 67% deuterium incorporations into 4a for the H
O (1:1, v/v) and D O incubations, respectively. Deuterium
incorporations were less than 100% in pure D O likely due
O in cells. H NMR (Figure 2, Table 2)
analysis indicated that the 1H signal for H
-2′ at δ 5.0
decreased as the concentration of D O in the incubation
increased. In deuterated 4a , a second doublet at δ 5.6 was
observed δ 0.01 upfield from the signal for H -2′ of 4. This
2
O-
D
2
2
158.3
2
3′
4
5
6
2
′
6.76 (d, 8.6)
7.21 (d, 8.4)
116.1
128.4
134.3
128.4
116.1
85.6
1′, 2′, 4′, 6′
2, 1′, 2′, 3′, 4′, 5′, 6′
2
1
to dilution by H
2
′
′
′
b
7.21 (d, 8.4)
6.76 (d, 8.6)
5.58 (dd, 8.8, 8.8)
2, 1′, 2′, 3′, 4′, 5′, 6′
1′, 2′, 4′, 6′
3, 7a, 3′, 4′, 5′
2, 3a, 4, 7a, 4′
2, 3a, 4, 7a, 4′
2
3 (Ha-3) 3.47 (dd, 9.1,15.7)
3
39.8
39.8
a
(Hb-3) 3.08 (dd, 8.5,15.7)
signal was in proportion to the amount of deuterium
incorporated into 4. Furthermore, the signal for H-1′
became a broad doublet (J ) 17.7 Hz), indicating coupling
3
4
5
6
7
7a
a
129.0
113.1
152.4
115.1
109.9
154.2
6.66 (br d, 2.4)
3a, 5, 6, 7a
with H
a b
-2′ but loss of coupling with H -2′. These results
6.54 (dd, 2.5,8.0)
6.57 (d, 8.5)
4, 5, 7a
3a, 5, 7a
indicated the specific incorporation of a single deuterium
1
atom into 4a at position H
deuterium incorporations of 29% and 76% for H
1:1, v/v) and 100% D O incubations, respectively.
The results of the D O-incorporation experiments sug-
b
-2′. H NMR analysis indicated
2
O-D
2
O
(
2
Isola tion a n d Ch a r a cter iza tion of a 4-Vin ylp h en ol
Dim er (11). The R 0.55 product (11, 5% yield by TLC,
2
f
gest a nonoxidative decarboxylation mechanism for the
formation of 4 by B. megaterium (Figure 1). Enzymatic
tautomerization of 1 would give a doubly vinylogous â-keto
acid, which nonenzymatically decarboxylates to 4. An
alternative mechanism for forming 4a , involving enzymatic
nucleophilic attack at C-3 of 111 and subsequent decar-
boxylation, cannot be differentiated by these experiments.
system A) was isolated from culture supernatants of B.
megaterium 24 h after substrate addition. HREIMS gave
an exact mass of 228.0802 for C14
12 3
H O , indicating a
product formed by coupling of 4 with a second six-carbon
1
moiety. By H NMR (Table 3) an AB system of doublets at
δ 7.21 and 6.76 indicated a p-substituted aromatic ring,
while a m-substituted aromatic ring gave o-coupling of the

1
410 J ournal of Natural Products, 2001, Vol. 64, No. 11
Torres y Torres and Rosazza
F igu r e 3. Conversion of 4-vinylphenol (4) and hydroquinone (7) by whole cell cultures of B. megaterium to the modified dimer of 4-vinylphenol
11).
(
Ta ble 4. NMR Data (methanol-d4) for
-[(E)-2-Carboxyethenyl]-2,3-dihydro-2-(4-hydroxyphenyl)-3-
benzofurancarboxylic Acid (15) Isolated from Curvularia lunata
1H signal at δ 6.54 (dd) with that at δ 6.57 and m-coupling
with that at δ 6.66. A pair of double doublets at δ 3.47 and
5
3
.08 indicated coupling between geminal methylene pro-
correlations
1
tons (J ) 15.7 Hz) that were vicinally coupled with a
H
signal at δ 5.58 (dd, J ) 8.8, 8.8 Hz). 1 C NMR indicated
3
HMBC
(C#)
NOESY
(H#)
position 1H (δ, J in Hz) 13C (δ)
that among 12 aromatic carbons, three at δ 158.3 (C-1′),
1
54.2 (C-7a), and 152.4 (C-5) were oxygen-substituted, two
2
3
3a
4
5
5.99 (d, 7.3)
4.26 (d, 7.3)
89.0 3, 7a, 1′, 2′, 6′, 1′′ 3, 2′, 6′
57.9 2, 3a, 4, 7a, 1′
128.6
2, 4, 2′, 6′
at δ 134.3 (C-4′) and 129.0 (C-3a) were carbon-substituted,
1
3
and the remaining aromatic carbons were methines.
C
7.66 (br d)
125.4 3, 6, 7a, 1′′
128.6
3, 2′′
signals at δ 128.4 (C-3′, C-5′) and 116.1 (C-2′, C-6′)
corresponded to carbons of the p-substituted aromatic ring,
while those at δ 115.1 (C-6), 113.1 (C-4), and 109.9 (C-7)
composed the m-substituted ring. A nonaromatic oxygen-
ated carbon at δ 85.6 (C-2) and a methylene carbon at δ
6
7.51 (dd, 1.5,8.4) 131.2 4, 7a, 1′′
7, 1′′, 2′′
7
7a
6.88 (d, 8.4)
109.3 3a, 5, 7a
162.4
6
1
2
3
4
′
′
′
′
132.8
7.21 (d, 8.4)
6.79 (d, 8.6)
128.0 2, 2′, 3′, 4′, 5′, 6′ 2, 3, 3′
116.0 1′, 3′, 4′, 5′
158.4
1
13
3
9.8 (C-3) were also detected. H and C NMR showed that
2, 2′
the vinyl group of 4 was absent in the metabolite and that
it contained a 2,3-dihydrobenzofuran ring.17,18 Key HMBC
data (Table 3) supported the presence of the 2,3-dihy-
drobenzofuran structure with substitution at positions 2
and 5. NMR analysis confirmed the structure of 11 as
5′
6′
6.79 (d, 8.6)
7.21 (d, 8.4)
7.65 (d, 15.7)
6.34 (d, 15.7)
116.0 1′, 3′, 4′, 5′
128.0 2, 2′, 3′, 4′, 5′, 6′ 2, 5′
146.0 4, 6, 2′′, 3′′
116.1 5, 3′′
171.0
2, 6′
1
2
3
1
′′
′′
′′
′′′
6, 2′′
4, 6, 1′′
4
-(2,3-dihydro-5-hydroxy-2-benzofuranyl)phenol, a previ-
175.5
ously unknown compound. The specific optical rotation of
0
° for 11 showed that the dimer was a (()-(2R,2S)-
1
TLC (system A, R
f
0.42), and H NMR and EIMS compari-
racemate.
son with data for authentic 2.
On the basis of its structure, 11 can form by a 1:1
dimerization of 4 and hydroquinone (7) (Figure 3). The
origin of hydroquinone (7) in the biotransformation product
Although 15 was mentioned once as a synthetic analogue
of a product from barley, no structural data have ever been
given.24 Negative-ion HRFABMS gave C18
H
13
O
6
(m/z
1
1 is unknown. It is possible that 7 is derived by vinyl-
3
25.0723), indicating a metabolite mass approximately
1
9
group elimination from 4, or it may be a biosynthetic
product of B. megaterium. Similar oxidative couplings have
been proposed for the dimerization of ferulic acid. The
final aromatization step is accompanied by 2,3-dihydroben-
zofuran ring formation giving a 1:1 racemate of (2R)- and
1
twice that of 1. H NMR (Table 4) supported a three-ring
structure similar to that of 11. One p-disubstituted aro-
matic ring was indicated by an AB system (o-coupled
doublets at δ 7.21 and 6.79), and a m-substituted aromatic
2
0
1
ring was indicated by an ABX system, for which the H
(2S)-(11). The dimer 11 was prepared biomimetically by
signal at δ 7.51 (dd) was o-coupled to that at δ 6.88 (d)
and m-coupled to that at δ 7.66 (d). trans-Coupling of
signals at δ 7.65 (d, J ) 15.7 Hz) and 6.34 (d, J ) 15.7 Hz)
identified an E-substituted vinyl group. Vicinal protons at
δ 5.99 (d, J ) 7.3 Hz) and 4.26 (d, J ) 7.3 Hz) provided
K
3
Fe(CN) oxidation of a mixture of 4-vinylphenol (4) and
6
2
1
hydroquinone (7) in chloroform and water. Pure 11 gave
HREIMS and H NMR data identical to those for 11
isolated from B. megaterium. The specific optical rotation
of 0° indicated that synthetic 11 was also a (()-(2R,2S)-
racemate.
1
2
0,25-27 13
strong evidence for a 2,3-dihydrobenzofuran ring.
C
NMR signals (Table 4) at δ 175.5 (C-1′′′) and 171.0 (C-3′′)
confirmed the presence of two carboxylic acid groups.
Signals at δ 162.4 (C-7a) and 158.4 (C-4′) showed two
oxygen-substituted aromatics and three carbon-substituted
aromatics δ 132.8 (C-1′), 128.6 (for C-3a and C-5), and the
remaining aromatic carbons were methines. Signals at δ
Proposed initial oxidative steps of such a pathway are
3 6
supported by the K Fe(CN)
-mediated synthesis of 11.2
0,22
The exact enzymatic process by which B. megaterium
catalyzes the oxidation reaction remains unknown. B.
megaterium contains a well-known cytochrome P450 en-
zyme system that could be capable of catalyzing such a
process.23
1
28.0 (C-2′, C-6′) and 116.0 (C-3′, C-5′) indicated a p-
substituted aromatic ring, while those at δ 109.3 (C-7),
125.4 (C-4), and 131.2 (C-6) composed the aromatic ring
with three substitutions. Carbons of an acrylic acid side
chain were identified by signals at δ 171.0 (C-3′′), 146.0
(C-1′′), and 116.1 (C-2′′), and two methine carbons at δ 57.9
(C-3) and 89.0 (C-2) were also observed. Key HMBC and
Meta bolism of 1 by C. lu n a ta . C. lunata transformed
to a new metabolite (15) (17% yield) and 2 (10% yield)
1
within 6 h. A preparative-scale C. lunata culture gave 31
mg of 2 and 110 mg of 15 from culture filtrates. The
structure of 2 as 4-hydroxybenzoic acid was confirmed by

Microbial Transformations of p-Coumaric Acid
J ournal of Natural Products, 2001, Vol. 64, No. 11 1411
NOESY data (Table 4) supported the presence of the 2,3-
dihydrobenzofuran structure with substitution at positions
1
13
2
, 3, and 5. The H signals for H-2 and H-3 and C signals
for C-2 and C-3 were very similar to those for the dehy-
drodimers of ferulic acid2
5,26
and the methyl ester of 1,
27
confirming the presence of the 2,3-dihydrobenzofuran ring
and the phenol substitution at C-2. Therefore, the structure
of the R
dihydro-2-(4-hydroxyphenyl)-3-benzofurancarboxylic acid
15).
The relative configurations of 2,3-dihydrobenzofurans
f
0.30 product was 5-[(E)-2-carboxyethenyl]-2,3-
(
1
about H-2 and H-3 can be determined by H NMR. In the
cis-configuration, H-2 is shifted downfield by approximately
0
.5 ppm vs trans H-2.28
3 6
K Fe(CN) -mediated phenol cou-
pling of the methyl ester of 1 gave the (()-trans dimethyl
ester of 15.2 H NMR and HRFABMS confirmed that our
synthetic dimer and 15 isolated from C. lunata were
identical. While the specific optical rotation of 0° indicated
that synthetic 15 was a racemate of (2R,3R)/(2S,3S)-trans
diastereomers, 15 isolated from C. lunata gave a specific
rotation of +3.8°, indicating a degree of stereospecificity
in the biotransformation reaction. Compound 15 is very
9 1
F igu r e 4. Proposed mechanism for the formation of the p-coumaric
acid dimer (15) from p-coumaric acid (1) by whole cell cultures of C.
lunata.
similar in structure to dimers of p-coumaryl alcohols and
ytruxillic acid and its cyclobutane stereoisomers (from
various grass cell walls),2 and 4-hydroxy-3-(4′-cinnamy-
loxy)cinnamic acid, the product of p-coumaric acid coupling
by an extracellular laccase from the basidiomycete Tram-
etes versicolor.37 Further investigation into the enzymes
involved in the biotransformation of p-coumaric acid by C.
lunata would be interesting, especially since the product
is not a racemate.
ferulic acid formed in lignin and lignans.2
5,30
All of the 2,3-
dihydrobenzofuran phenol dimers formed by in vitro oxida-
tive phenol couplings by peroxidases or chemical oxidations
have the (()-trans relative configuration between H-2 and
H-3.1
7,20,25,27,30,31
However, lignans are formed in vivo by
3
0
stereospecific phenol coupling reactions, as in (+)-geodin
from Aspergillus terreus.32 Conclusive determination of the
absolute configuration of 15 will require X-ray crystal-
lography, which has successfully proven the absolute
configuration of other phenolic 2,3-dihydrobenzofuran
Exp er im en ta l Section
1
Gen er a l Exp er im en ta l P r oced u r es. One-dimensional H
2
0,29,33
13
dimers, including that of dehydrodiferulate.
and C NMR spectra were recorded with a Bruker WM-360
MHz, a Bruker DRX-400 MHz, or a Bruker AMX-600 MHz
instrument (Karlsruhe, Germany). Samples were analyzed by
heteronuclear multiple bond correlation (HMBC) or by nuclear
Overhauser effect spectroscopy (NOESY) experiments by the
Bruker AMX-600 spectrometer. Chemical shifts are recorded
in δ value (ppm) downfield from TMS. Coupling constants (J
values) are recorded in hertz. Electron-impact mass spectrom-
etry (EIMS) was performed with either a Trio-1 mass spec-
trometer (VG Analytical, Manchester, England) or a Voyager
mass spectrometer (ThermoQuest, Manchester, England). For
direct inlet probe mass spectrometry (DIPMS), the ionization
temperature of the probe was held at 30 °C for 1 min and
increased linearly over 2 min to 320 °C. Gas chromatography-
mass spectrometry (GC/MS) was carried out by the Trio-1 mass
spectrometer at 70 eV connected to a Hewlett-Packard 5890
A gas chromatograph (Palo Alto, CA) using a DB-1 methyl-
silicone column (15 × 0.32 mm i.d., J & W Scientific, Folsom,
CA). The initial analysis temperature of 50 °C was increased
at a rate of 20 °C/min to 250 °C and held at this temperature.
Negative ion fast atom bombardment mass spectrometry
C. lu n a ta En zym es. Assays were conducted with
culture filtrates and cell-free extracts of C. lunata for
laccase and peroxidase activities. Laccases are copper
oxidases that oxidize phenols through the reduction of
molecular O
Peroxidases also oxidize phenols by H
2
to H
2
O by single electron transfer reactions.
-dependent one-
2
O
2
electron oxidation. Oxidation of syringaldazine indicates
laccase activity.34 C. lunata culture filtrates rapidly oxi-
dized syringaldazine to the deeply purple syringaldazine
quinone,34 while cell-free extracts were only weakly positive
for laccase. Peroxidase activity was present in both the cell-
free extract (35 nmol/min) and culture filtrates (23 nmol/
min), demonstrating that either laccase or peroxidase could
catalyze the dimerization of 1 to 15.
The presence of laccase35 and peroxidase36 activities in
C. lunata and the K
from 1 support an initial radical-based coupling process
Figure 4) involving phenoxy radicals (12), quinone me-
3 6
Fe(CN) -mediated synthesis of 15
(
thide, and semiquinone radicals (13) that couple to give a
mixture of (R)- and (S)-14. Subsequent cyclization would
give racemic trans-substituted dihydrobenzofurans 15.20
(
(
FABMS) was carried out with a ZAB-HF mass spectrometer
VG Analytical, Manchester, England). The ionizing matrix
was triethanolamine.
Caffeic acid (5), p-coumaric acid (1), deuterium oxide,
Microbial transformation of p-coumaric acid (1) afforded
4
-hydroxybenzoic acid (2), 4-vinylphenol (4), caffeic acid (5),
4-hydroxybenzoic acid (2), potassium(III) ferricyanide (K Fe-
3
protocatechuic acid (6), and other products by several
microorganisms for which these conversions had not been
previously reported. Additionally, dimers 11 and 15 from
B. megaterium and C. lunata are new and previously
unidentified biotransformation products. The isolation of
6
(CN) ), protocatechuic acid (6), pyrogallol, syringaldazine, and
tetramethylsilane (TMS) were purchased from Aldrich (Mil-
waukee, WI). Phenylmethylsulfonylfluoride (PMSF) was ob-
tained from Amresco (Solon, OH), dithiothreitol (DTT) was
purchased from GIBCO Laboratories (Grand Island, NJ ), and
NMR solvents were purchased from Isotec, Inc. (Miamisburg,
OH). 4-Vinylphenol [4, a 10% solution (w/w in propylene
glycol)] was purchased from Lancaster, Inc. (Windham, NH),
and 100% EtOH was obtained from Pharmco Products (Brook-
field, CT). All other chemicals were purchased from Fisher
Chemicals (Fair Lawn, NJ ).
1
1 and 15 from microorganisms demonstrates the microbial
formation of phenolic acid dimers similar to those found
in plant cell walls, lignin, and lignans. Only two dimers of
1
, both of different structures from 15, have been previously
characterized. These include the photodimer 4,4′-dihydrox-

1
412 J ournal of Natural Products, 2001, Vol. 64, No. 11
Torres y Torres and Rosazza
Compounds were separated by thin-layer chromatography
TLC) on either Merck silica gel GF254 spread over glass plates
with a Quickfit Industries (London, England) spreader (0.25
mm thickness, activated at 120 °C for 20 min) or aluminum
foil-backed (Alltech) Kieselgel 60 F254 plates. Plates were
addition, acidified to pH 2.0, and extracted with 1 mL of
EtOAc-propanol (9:1, v/v). The organic layer was separated
by centrifugation to 1200g in a desktop centrifuge, and 40 µL
samples were spotted onto TLC plates and developed in system
A.
(
developed by one of the following systems (v/v): (A) CH
MeOH-HCOOH (95:5:0.5), (B) C 14-EtOAc (1:1), (C) CH
Cl -MeOH-HCOOH (93:7:0.5). Compounds were visualized
by viewing the plates at 254 and 366 nm and by spraying with
diazotized sulfanilic acid reagent, which was prepared by
mixing (1:1, v/v) solution A [0.5% (w/v) sulfanilic acid in 2%
2
Cl
2
-
P r ep a r a tive Tr a n sfor m a tion of p-Cou m a r ic Acid (1)
to 4-Vin ylp h en ol (4) by B. m ega ter iu m . After incubation
of stage II cultures (10 1 L DeLong flasks, 200 mL medium
each) for 24 h at 28 °C, 1 was added (0.8 mg/mL). Controls
consisted of 25 mL of medium in 125 mL flasks containing
either 1 (0.8 mg/mL), 4-vinylphenol (4, 1 mg/mL), or no
substrate incubated under the same conditions. Samples (4
mL) from each flask were taken and assayed by TLC (system
6
H
2
-
2
HCl] with solution B [0.5% (w/v) NaNO
the plate, followed by spraying with solution C [5% NaOH in
0% EtOH] and heating. Optical rotations were measured with
2 2
in H O] and heating
5
f
A) to show 4 (R 0.70, red) in estimated 40% and 50% yields
a J ASCO P-1020 polarimeter (Kyoto, J apan), and UV spectra
were obtained with a UV-2101PC scanning spectrophotometer
after 6 and 24 h, respectively.
After 24 h, the contents of five flasks were centrifuged at
10 000g for 10 min. Pellets containing only traces of 1 or 4
were discarded. The supernatant (1 L) was adjusted to pH 2.0
with 6 N HCl and extracted with three 500 mL volumes of
EtOAc. The organic layers were pooled, dried over Na SO ,
2 4
filtered through sintered glass, and evaporated to a brown oil
(1.5 g). A 0.5 g sample of oil was dissolved in 25 mL of EtOAc
(Shimadzu). Melting points were obtained with a Mel-Temp
apparatus.
Microbial cells were broken by an Aminco French Pressure
Cell (American Instrument Co., Silver Spring, MD). Whole and
homogenized cells were centrifuged with either a Sorvall RC-5
superspeed refrigerated centrifuge (Dupont Instruments, New-
ton, CT), an Eppendorf 5415C microcentrifuge (Germany), or
an L8-55 preparative ultracentrifuge (Beckman Instruments,
Palo Alto, CA).
and extracted with three 25 mL volumes of 5% (w/v) NaHCO
The organic layer was dried over Na SO and concentrated to
afford 85 mg of red oil that was resolved over 11 g of 60-200
3
.
2
4
For HPLC, a Shimadzu LC-6A dual pumping system was
connected to a Shimadzu SPD-6AV UV/vis detector. Samples
were introduced onto the column (Versapack C18 10µ, 250 mm
mesh silica gel (1.7 × 10.0 cm) with CHCl
3
-MeOH (95:5) to
provide 25 mg of 4-vinylphenol (4, 13%) after evaporation of
pooled fractions.
Meta bolite 4: off-white powder; H NMR (Me
1
×
4.6 mm, Alltech, Deerfield, IL) through a Shimadzu 7161
2
CO-d
6
, 360
injection port. Chromatograms were recorded and processed
by the Shimadzu ClassVP program. Chemicals were diluted
in Optima grade MeOH and injected in triplicate. C. lunata
culture samples were centrifuged at 14 000 rpm for 1 min in
an Eppendorf centrifuge. The supernatant was passed through
a 0.45 mm filter, diluted 1:1 (v/v) with Optima grade MeOH,
and injected in triplicate (20 µL each). Controls included
cultures without substrate and substrate added to uninocu-
lated medium. Standard curves were constructed by injecting
in triplicate at least three concentrations of each compound.
Culture samples were eluted isocratically with MeOH-2%
HCOOH, 1:1 (v/v), at 1 mL/min (265 nm). Retention volumes
MHz) δ 7.30 (2H, d, J ) 8.6 Hz, H-2, H-6), 6.81 (2H, d, J ) 8.6
Hz, H-3, H-5), 6.65 (1H, dd, J ) 10.8, 17.6 Hz, H-1′), 5.59 (1H,
dd, J ) 1.1, 17.6 Hz, H -2′), 5.03 (1H, dd, J ) 1.1, 10.8 Hz,
a
+
+
H -2′); EIMS (70 eV) m/z 121 [M + 1] (5%), 120 [M] (100%),
b
+
91 (36%), 65 [C H ] (8%); spectral data were indistinguishable
5 5
from that from a commercial standard of 4 and from the
literature.39
2
Con ver sion of 1 t o 4 (Vin ylp h en ol) a n d 4a in D O.
Stage I and II cultures of B. megaterium (25 mL of medium in
125 mL flasks) were incubated at 28 °C as described above.
After 24 h, eight cultures were combined and centrifuged at
10 000g for 15 min. The supernatant was decanted, and the
(R
v
, mL) of eluted compounds were 5.0 for 4-hydroxybenzoic
2 4
pellet was washed with 0.1 M KH PO , pH 7.0 (120 mL), and
acid (2), 6.4 for 1, and 16.4 for 15.
recentrifuged at 10 000g for 15 min. The resulting pellet was
Micr oor ga n ism s. All microorganisms were maintained on
Sabouraud maltose agar except G. deliquescens (Czapek’s agar,
American Type Culture Collection medium no. 312). All
cultures were stored at 4 °C prior to use. Fifty-two microor-
ganisms of the following genera were used, including three
Aspergillus spp., nine Bacillus spp., one Caldariomyces sp.,
one Candida sp., seven Cunninghamella spp., one Curvularia
sp., one Cylindrocarpon sp., one Gliocladium sp., one Mem-
noniella sp., three Mucor spp., one Mycobacterium sp., four
Nocardia spp., three Penicillium spp., five Pseudomonas spp.,
one Rhizopus sp., one Rhodotorula sp., one Saccharomyces sp.,
one Sarcina sp., one Schizosaccharomyces sp., five Streptomy-
ces spp., and an unidentified soil microorganism.
divided into four equal portions, and each was resuspended
in 25 mL of one of the following solutions: (1) 0.1 M KH
pH 7.0; (2) 0.1 M KH PO , pH 7.0, 1 mg/mL p-coumaric acid
(1); (3) 0.1 M KH PO prepared in D O/H O (1:1, v/v), pH 7.0,
1 mg/mL 1; (4) 0.1 M KH PO prepared in D O, pH 7.0, 1 mg/
2 4
PO ,
2
4
2
4
2
2
2
4
2
mL 1. Suspended cells were incubated in 125 mL DeLong
flasks at 28 °C, 250 rpm. The reaction progress (solution 2)
was monitored by TLC system A, which indicated that 4 was
produced in nearly 100% yield after 6 h. After 6 h, cultures
were centrifuged at 10 000g for 15 min, and the supernatants
were extracted with 3 × 12 mL of EtOAc. After concentration,
3 mg (D
2
2 2
O-H O, 1:1, v/v) and 9 mg (D O) of residues were
isolated. These two residues were chromatographed separately
Scr een in g a n d An a lytica l Sca le Cu ltu r e Con d ition s.
over 60-200 mesh silica gel (8.0 × 0.6 cm, 2 mL/min) with
3
8
A two-stage fermentation protocol was used for screening and
preparative isolation of p-coumaric acid (1) metabolites. The
incubation medium consisted of 0.5% (w/v) yeast extract, 0.5%
6
C H14-EtOAc-HCOOH (75:25:0.5). Seven 0.5 mL fractions
from each column were pooled and evaporated to yield 3 mg
mixtures of 4 and 4a from D O-H O (1:1) and D O flasks. A
similar workup gave 6 mg of 4 from H O incubations. All
2
2
2
NaCl, 0.5% K
2
HPO
4
, 0.5% soybean meal, and 2% dextrose per
2
1
1
L of distilled water, adjusted to pH 7.0 with 6 N HCl. Stage
samples were subjected to low-resolution GC/MS and H NMR
spectral analyses (Table 2 and Figure 2).
I cultures were grown in 25 mL of sterile soybean meal-
glucose medium held in 125 mL stainless steel-capped DeLong
flasks and autoclaved at 121 °C for 15 min. Flasks were
incubated at 28 °C and 250 rpm on New Brunswick Scientific
Isola tion of (2R,2S)-4-(2,3-Dih yd r o-5-h yd r oxy-2-ben -
zofu r a n yl)p h en ol (11). After 24 h, stage II B. megaterium
cultures (400 mL, 25 mL in sixteen 125 mL flasks) received
4-vinylphenol (4, 1 mg/mL, 400 mg total). Controls included
cultures without 4 and uninoculated medium with 4 added.
(
New Brunswick, NJ ) G10, G25, or Innova 5000 Gyrotory
shakers. After 72 h, 25 mL stage II fermentations were started
in the same medium with a 10% inoculum of stage I culture.
Stage II cultures were incubated for 24 h, when p-coumaric
acid (1) was added to flasks to 1 mg/mL culture medium (250
µL of a 0.1 g/mL solution of 1 in EtOH). Controls included
uninoculated sterile medium containing 1 mg/mL 1 and
inoculated medium without substrate. Samples (4 mL) were
withdrawn for analysis 6, 24, 72, and 144 h after substrate
Dimer 11 (brown, R
estimated 5% yield in 24 h. Cultures were combined and
centrifuged (10 000g, 10 min), and 125 mL of 1 M NaHCO
was added to adjust 375 mL of the supernatant to pH 8.0,
Cl . Extracts
and concentrated to give 353 mg of a
f
0.55, TLC system A) was formed in an
3
before being extracted with 2 × 500 mL of CH
2
2
were dried over Na
yellow-red oil.
2 4
SO

Microbial Transformations of p-Coumaric Acid
J ournal of Natural Products, 2001, Vol. 64, No. 11 1413
The oil was chromatographed over 60-200 mesh silica gel
CH
2 2
Cl -MeOH. Fractions containing a tan solid 15 (34 mg,
25
(
26.5 × 2.3 cm) with 96:4 CH
2
Cl
2
-MeOH. Fractions were
0.10 mmol, 19% yield) gave [R]
D
0.0° (c 0.5, MeOH); UV, NMR
pooled and concentrated to give 31 mg of a yellow residue,
which was separated over a second silica gel column (11.5 ×
(Table 4) and MS spectral properties essentially identical to
those of 15 isolated from C. lunata; HRFABMS gave m/z
325.0710.
1
1
.5 cm) with C
6
H
14-EtOAc (60:40) to yield 1.5 mg (0.4%) of
1.
An a lysis of C. lu n a ta En zym es. Five C. lunata stage II
cultures (each with 200 mL Iowa medium/1 L DeLong flask)
were incubated for 24 h (28 °C). One flask received 1 (1 mg/
mL). Twenty-four hours later, the substrate-containing flask
was assayed for conversion products of 1 (TLC system A) to
confirm the presence of metabolites. After 48 h, the other four
stage II culture flasks were harvested by filtration through
cheesecloth. Filtered mycelia were washed with 350 mL of pH
5.8, 10 mM phosphate buffer containing 0.5% NaCl, and
combined mycelia were frozen at -70 °C. Culture filtrates were
stored at 4 °C. Mycelium (15 g) was thawed and suspended in
45 mL of sodium phosphate buffer (30 mM, pH 8.1) containing
2 mM DTT, 2 mM PMSF, and 20% glycerol (v/v),41 and passed
six times through a French press (1100 psig, 18 000 psi).
Homogenized cells were centrifuged at 25 000g for 30 min at
4 °C to produce a cell-free extract. Laccase activity was
qualitatively determined in 0.5 mL of culture filtrate or cell-
free extract by adding 10 µL of syringaldazine (4.44 mM in
MeOH), mixing vials by inversion, and viewing the develop-
ment of a deep pink-purple color over 2-10 min. Controls
contained only MeOH.
Peroxidase activity was assayed by monitoring the increase
in absorbance at 420 nm at 20 °C for 5 min. Peroxidase assay
mixtures contained 42 mM pyrogallol, and cell-free extract,
or culture filtrate in 3 mL of sodium phosphate buffer (0.1 M,
pH 5.8) and 160 µL of H
160 µL of buffer in the blank.
Met a bolit e 11: off-white powder; [R]2
5
D
0.0° (c 0.05,
1
13
MeOH); UV (MeOH) λmax 226, 283, 300 nm; H and C NMR,
+
HMBC (Table 3); EIMS (70 eV) m/z 228 [M] (100), 211 [M -
+
+
+
6 5
H ] (14);
OH] (13), 181 (12), 134 [M - C
6
H
6
O] (5), 77 [C
, 228.0786).
Oxid a tion of 4 a n d
4-Vinylphenol (4, 121 mg, 1.0 mmol)
and 7 (111 mg, 1.0 mmol) in 20 mL of
acetone were combined, and 97 mL of an aqueous solution of
Fe(CN) (578 mg, 1.8 mmol) and Na CO (3 g, 27.4 mmol)
were added. The resulting mixture was stirred under N in
HREIMS m/z 228.0802 (calcd for C14
12 3
H O
P r ep a r a tion of 11 by K F e(CN)
3
6
2
1,29
Hyd r oqu in on e (7).
in 200 mL of CHCl
3
K
3
6
2
3
2
the dark and monitored by TLC (system B) to show an
estimated 50% yield of 11 in 22 h. Chloroform and aqueous
layers were separated, and the aqueous layer was extracted
with 3 × 48 mL volumes of EtOAc. The organic layers were
combined, dried over Na
of dark brown oil.
2 4
SO , and evaporated to give 32 mg
The oil was resolved over a 5 g, 40 µm SiO
2
column (13.5
14-EtOAc (1 mL fractions). After
3
4
cm × 1.1 cm) with 4:1 C
6
H
evaporation, one fraction contained 9 mg of 11 (4% yield).
Oxid a tion p r od u ct 11: white powder; mp 161-163.5 °C
2
5
42
(
(
decomposition, corrected); [R]
D
0.0° (c 0.5, MeOH); UV
MeOH) λmax (log ꢀ) 228 (4.07), 286 (3.37), 302 (3.52) nm; NMR
and MS spectral analyses were essentially identical to those
for 11 isolated from B. megaterium (Table 3).
P r ep a r a tive Tr a n sfor m a tion of 1 by C. lu n a ta . Six 24-
h-old stage II cultures (200 mL soybean meal-glucose medium
in 1 L flasks) received 1.2 g of p-coumaric acid (1, 1.0 mg/mL).
After 24 h, TLC system C indicated the presence of two new
2 2 2 2
O (8 mM). H O was replaced by
Ack n ow led gm en t. We thank L. Dostal for assistance with
screening and J . Snyder and D. Herschberger of the University
of Iowa NMR Facility and Mass Spectrometry Facilities for
technical assistance. J .T.T. acknowledges financial support
through a National Institutes of Health Training Grant/Center
for Biocatalysis and Bioprocessing Fellowship in Biotechnology
f f
metabolites, 15 (R 0.35, brown) and 2 (R 0.47, yellow), which
were present in approximately 20% and 10% yields, respec-
tively. Cultures were harvested by filtration through cheese-
cloth, and the filtrate was passed through Whatman No. 1
filter paper. The culture filtrate was adjusted to pH 2.0 with
(GM# 08365) and the American Foundation for Pharmaceuti-
6
N HCl and extracted with 3 × 600 mL of 9:1 EtOAc-
cal Education for a Predoctoral Fellowship. We also thank the
USDA through the Biotechnology Byproducts Consortium for
financial support.
propanol to give 1 g of red oil after evaporation.
The red oil was resolved over 100 g of 40 µm silica gel (32.0
×
2 2
3.5 cm), eluted with CH Cl -MeOH (9:1). Similar fractions
were pooled to give 203 mg of a dark orange oil containing 2
and 232 mg of impure 15. Chromatographic resolution of the
Refer en ces a n d Notes
(
(
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oil over 20 g of 40 µm silica gel (17.4 × 1.9 cm) with CHCl
3
-
MeOH (95:5) gave 31 mg (0.22 mmol, 3% yield) of 4-hydroxy-
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1
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) 253 (4.10) nm; H NMR and EIMS analyses were in
(
4
0
agreement with data for authentic 2; HREIMS m/z 138.0321
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Pure 15 was obtained by further resolution of the impure
8
(
7
H
6
O
3
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2
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2
5
tion, corrected); [R]
D
+3.8° (c 1.0, MeOH); UV (MeOH) λmax
1 13
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(
log ꢀ) 227 (4.27), 300 (4.21), 317 (4.24); H, C NMR, HMBC,
NOESY (Table 4); EIMS (70 eV) m/z 326 [M] (0.3%), 282 [M
] (13), 264 [M - CO
15 (13); FABMS m/z 325 [M - H] (48), 281 [M - H - CO
100), 255 [M - H - C
29); HRFABMS m/z 325.0723 (calcd for C18
P r ep a r a tion of 15 by K F e(CN) Oxid a tion of 1.
p-Coumaric acid (1, 179 mg, 1.1 mmol) in 30 mL of acetone
was added to 270 mL of CHCl . K Fe(CN) (450 mg, 1.37 mmol)
and Na CO (2.25 g, 21.0 mmol) were dissolved in 75 mL of
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+
(
+
+
+
-
1
(
(
CO
2
2
, H
2
O] (4), 238 [M - 2CO
2
] (100),
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-
-
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2
]
]
-
2 2 2
H CO ] (13), 237 [M - H - 2CO
-
21.
2
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H O , 325.0712).
1
30, 425-433.
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1,29
3
6
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(
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f
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2 4
SO and concentrated to 214 mg
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414 J ournal of Natural Products, 2001, Vol. 64, No. 11
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