Hydroxylation of ionized aromatics including carboxylic acid or amine using recombinant Streptomyces lividans cells expressing modified biphenyl dioxygenase genes

Article abstract of DOI:10.1016/S0040-4020(03)00180-7

The bphA1(2072)A2A3A4 gene cluster codes for a shuffled biphenyl dioxygenase holoenzyme with broad substrate specificity. These bphA1(2072)A2A3A4 genes were expressed in the actinomycetes Streptomyces lividans using a thiostrepton-inducible promoter P_tipA. Biotransformation experiments of various aromatics including carboxylic acid or amine in their molecular structure, such as 1-naphthoic acid, 2-(1-naphthyl)acetic acid, diphenylamine, and 1-benzyl-4-piperidone, were performed using the recombinant S.lividans cells. These ionized aromatics were converted to the corresponding 1,2-dihydrodiol, mono- or tri-hydroxy forms in 48h. The structure of the converted products was determined by their EI-MS, ¹H- and ¹³C NMR analysis, and several products were found to be novel compounds.

Full text of DOI:10.1016/S0040-4020(03)00180-7

Tetrahedron 59 (2003) 1895–1900
Hydroxylation of ionized aromatics including carboxylic acid or
amine using recombinant Streptomyces lividans cells expressing
modified biphenyl dioxygenase genes
a
b
c
c
Kazutoshi Shindo,a Ryoko Nakamura, Ikuko Chinda, Yasuo Ohnishi, Sueharu Horinouchi,
Haruko Takahashi,d Kazuo Iguchi,d Shigeaki Harayama,e Kensuke Furukawaf
and Norihiko Misawab,e
,
*
aDepartment of Food and Nutrition, Japan Women’s University, 2-8-1, Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
bCentral Laboratories for Key Technology, Kirin Brewery Co. Ltd, 1-13-5, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
cDepartment of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, Japan
dTokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
eMarine Biotechnology Institute, 3-75-1, Heita, Kamaishi 026-0001, Japan
fGraduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan
Received 31 August 2002; accepted 27 January 2003
Abstract—The bphA1(2072)A2A3A4 gene cluster codes for a shuffled biphenyl dioxygenase holoenzyme with broad substrate specificity.
These bphA1(2072)A2A3A4 genes were expressed in the actinomycetes Streptomyces lividans using a thiostrepton-inducible promoter PtipA
.
Biotransformation experiments of various aromatics including carboxylic acid or amine in their molecular structure, such as 1-naphthoic
acid, 2-(1-naphthyl)acetic acid, diphenylamine, and 1-benzyl-4-piperidone, were performed using the recombinant S. lividans cells. These
ionized aromatics were converted to the corresponding 1,2-dihydrodiol, mono- or tri-hydroxy forms in 48 h. The structure of the converted
1
products was determined by their EI-MS, H- and 13C NMR analysis, and several products were found to be novel compounds. q 2003
Elsevier Science Ltd. All rights reserved.
1. Introduction
fully by this recombinant Escherichia coli strain carrying
the modified biphenyl dioxygenase genes
Degradation of the environmental pollutants, polychlori-
nated biphenyls (PCBs) is initiated enzymatically by the
action of biphenyl dioxygenase.1 This enzyme is a multi-
component enzyme that consists of BphA1, BphA2 (large
and small subunits of iron-sulfur protein, respectively),
BphA3 (ferredoxin), and BphA4 (ferredoxin reductase).
[bphA1(2072)A2A3A4].3 The 1,2-dihydrodiols produced
including heteroaromatics have important industrial poten-
tial as versatile starting materials for the enantioselective
chemical synthesis of biologically active organic molecules,
such as therapeutic agents that include heterocycles in
their molecular structure. In addition this study also
illustrated that the use of biphenyl dioxygenase could
mediate the effective synthesis of high value organic
molecules.
Recently, modified bphA1 genes have been generated by
DNA shuffling using the bphA1 genes derived from the
gram-negative bacteria Pseudomonas pseudoalcaligenes
KF707 and Burkholderia cepacia LB400.2 One of the
shuffled genes, bphA1(2072), has been shown to mediate a
broad substrate specificity, when expressed in combination
with bphA2A3A4 from P. pseudoalcaligenes.3 Various
molecular species, in which heterocyclic aromatics are
linked with phenyl or benzyl groups, were shown to be
converted to their corresponding 1,2-dihydrodiols success-
So far, the biphenyl dioxygenase-mediated transformation
of aromatics to cis-diols has mainly been studied for
bioremediation of environmental pollutants. Plasmid,
pIJ6021-bphA1(2072)A2A3A4, for the expression of the
modified biphenyl dioxygenase genes in the gram-positive,
soil-inhabiting, filamentous bacterium Streptomyces
lividans has also been constructed using a thiostrepton-
inducible promoter PtipA on a high-copy-number vector
pIJ6021.4 The S. lividans transformant carrying this plasmid
was able to catalyze the same conversions as those by the
E. coli transformant at the same or improved efficiency
(Y. Ohnishi and S. Horinouchi, unpublished results).
Keywords: biphenyl dioxygenase; biotransformation; aromatics;
Streptomyces lividans.
*
Corresponding author. Tel.: þ81-3-5981-3433; fax: þ81-3-5981-3426;
e-mail: kshindo@fc.jwu.ac.jp
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00180-7

1896
K. Shindo et al. / Tetrahedron 59 (2003) 1895–1900
Ionized aromatic molecules including carboxylic acid or
amine moieties in their molecular structure have frequently
been used as building blocks for the chemical synthesis of
various therapeutic agents and agrochemicals.5 Biological
modification of these molecules through hydroxylation
reactions is a promising way to extend a variety of
biologically active chemicals. In this paper we describe
hydroxylation of ionized aromatic molecules, such as
1-naphthoic acid, 2-(1-naphthyl)acetic acid, diphenylamine,
1-benzyl-4-piperidone, benzyl-carbamic acid tert-butyl
ester and phenyl-carbamic acid tert-butyl ester, through
the modified biphenyl dioxygenase-carrying S. lividans
cells.
2.2. Biotransformation of diphenylamine and 1-benzyl-
4-piperidone
Aromatics including secondary and tertiary amine (diphenyl
amine and 1-benzyl-4-piperidone, respectively) were used
as substrate for biotransformation experiments. Both the
compounds were converted to the corresponding mono- or
tri-hydroxy forms, or 1,2-dihydrodiol form with high
efficiency (60–80%) by the recombinant S. lividans cells.
The recombinant E. coli was also able to convert these
aromatic molecules.
2.2.1. Diphenylamine. Diphenylamine was converted to
three products by the S. lividans transformant. The molecu-
lar formulas of the two products (5, 6) were determined to be
1
C12H11NO by HR-EIMS. Analysis by H–13C COSY and
2. Results
DQF COSY spectra revealed one phenolic OH was replaced
in the benzene ring of products 5 and 6. Observation of
visinal spin networks from H-3 (d 6.88) to H-5 (d 7.08)
and H-20 (d 6.68) to H-60 (d 6.68) confirmed that 5 was
2-anilonophenol10 (Fig. 1). The symmetrical 1H NMR
spectra revealed that 6 was 4-anilonophenol11 (Fig. 1).
2.1. Biotransformation of 1-naphthoic acid and 2-(1-
naphthyl)acetic acid
Aromatics including the carboxylic acids (1-naphthoic acid
or 2-(1-naphthyl)acetic acid) were converted to the mono-
hydroxy derivatives. A 40–60% conversion was achieved
from each substrate using the S. lividans cells expressing the
modified biphenyl dioxygenase genes (plasmid, pIJ6021-
bphA1(2072)A2A3A4). In contrast, the recombinant E. coli
cells expressing the same modified biphenyl dioxygenase
genes (plasmid, pKF2072) were not able to convert these
aromatic molecules.
The molecular formula of the remaining product (7) was
determined to be C6H11NO3 by HR-EIMS. Analysis by
1H–13C COSY and DQF COSY spectra revealed three
phenolic OHs were replaced in the benzene rings. The
positions of the OH moieties were determined to be C-2,
C-30 and C-40 by the observation of visinal spin networks
from H-3 (d 7.32) to H-6 (d 7.77), between H-5 (d 7.42) and
H-6 (d 6.84) as well as meta coupling between H-20 (d 6.31)
and H-60 (d 6.84). From these findings, product 7 was
determined as 4-(2-hydroxyanilino)-1,2-benzenediol
(Fig. 1). 7 was a novel compound.
2.1.1. 1-Naphthoic acid. 1-Naphthoic acid was converted to
two products by the S. lividans transformant. The molecular
formulas of both the products (1, 2) were determined to be
1
C11H8O3 by HR-EIMS. Analysis by H–13C COSY and
DQF COSY spectra revealed one phenolic OH was replaced
in the naphthalene ring of products 1 and 2. In the HMBC
2.2.2. 1-Benzyl-4-piperidone. 1-Benzyl-4-piperidone was
converted to one product by the S. lividans transformant.
The molecular formula of the product (8) was determined to
1
spectrum of 1, H–13C long range couplings from H-2 (d
8.12) to carbonyl carbon (d 168.2) and C-4 (d 157.8)
were observed. Therefore, product 1 was identified to be
4-hydroxy-1-naphthoic acid6 (Fig. 1). Visinal sp2 spin
network of H-2 (d 8.07)–H-3 (d 7.48)–H-4 (d 8.37) in the
1
be C11H17NO3 by HR-EIMS. Analysis by H–13C COSY
and DQF COSY spectra showed that 8 was a dihydrodiol
derivative of the benzene ring. The 2,3-diol regiochemical
assignment was confirmed by the observation of sp2 visinal
spin network of H-4 (d 5.70)–H-5 (d 5.90)–H-6 (d 5.90).
Thus, product 8 was determined as 4-(2-hydroxyanilino)-
1,2-benzenediol (Fig. 1). The absolute stereochemistry of 8
was determined by 1H NMR analysis of diastereomeric
esters formed with (R)- and (S)-methoxy-(2-naphthyl)acetic
acid (2NMA). D(dR2dS) Values are summarized in Figure
2. The sign of Dd are systematically arranged right and left
sides to the 2NMA planes. From these results, the absolute
configuration of C-2 and C-3 in 8 were determined to be R
and S, respectively. 8 was also a novel compound.
1
DQF COSY spectrum and H–13C long range couplings
from H-2 to carbonyl carbon (d 168.8) and H-4 to C-5 (d
153.6) in the HMBC spectrum showed that product 2 was
5-hydroxy-1-naphthoic acid7 (Fig. 1). The yield of the
products purified is also shown in Figure 1.
2.1.2. 2-(1-Naphthyl)acetic acid. 2-(1-Naphthyl)acetic
acid was converted to two products by the S. lividans
transformant. The molecular formulas of both the products
(3, 4) were determined to be C12H10O3 by HR-EIMS.
Analysis by 1H–13C COSY and DQF COSY spectra
revealed one phenolic OH was replaced in the naphthalene
ring of products 3 and 4. In the HMBC spectrum of 3,
1H–13C long range couplings from H-10 (d 3.96) to C-2 (d
128.2) and H-2 (d 7.18) to C-4 (d 152.3) were observed.
Therefore, product 3 was identified to be 2-(4-hydroxy-1-
naphthyl)acetic acid8 (Fig. 1). Visinal sp2 spin network of
H-2 (d 7.39)–H-3 (d 7.39)–H-4 (d 8.16) in the DQF COSY
2.3. Biotransformation of benzyl-carbamic acid tert-
butylester and phenyl-carbamic acid tert-butylester
Benzylamine and aniline, which include an amino group
(primary amine) in their molecular structure, were not
converted by the recombinant S. lividans cells or by the
recombinant E. coli cells. This could be due to the luck of
their permeability into the cell or perhaps their affinity to the
iron-including active site of the enzyme. Therefore, the
amino groups of benzylamine and aniline were protected as
1
spectrum and H–13C long range couplings from H-2 to
C-10 (d 39.2) and H-4 to C-5 (d 153.0) in the HMBC
spectrum showed that product 4 was 2-(5-hydroxy-1-
naphthyl)acetic acid9 (Fig. 1).

K. Shindo et al. / Tetrahedron 59 (2003) 1895–1900
1897
Figure 1. Structures of products bioconverted from respective aromatic compounds. Percent value in the parentheses represents the yield of the products
purified.
tert-butyl carbamate by stirring with di-tert-butyl dicarbo-
nate [(t-BOC)2O] and NaOH in aqueous dioxane. Their
protected groups should easily be removed by acidic
treatment. The t-BOC derivatives synthesized were success-
fully converted to the corresponding 1,2-dihydrodiol or
monohydroxy forms with high efficiency (75–85%) by the
recombinant S. lividans cells. The recombinant E. coli was
also able to convert these aromatic molecules.
2.3.1. Benzyl-carbamic acid tert-butyl ester. Benzyl-
carbamic acid tert-butyl ester was converted to one product
by the S. lividans transformant. The molecular formula of

1898
K. Shindo et al. / Tetrahedron 59 (2003) 1895–1900
Enzyme-mediated introduction of (a) hydroxyl group(s) into
the benzene rings of various aromatic compounds including
carboxylic acid or amine moieties has been described in this
study. The hydroxylated metabolites are easily prepared
with high yields using the recombinant bacterial cells that
carry the modified biphenyl dioxygenase genes. The scale-
up experiments should be easy to do. For example, in the
case of 1-benzyl-4-piperidone as the substrate, the addition
of one gram to 1-liter culture gave 326 mg of the converted
compound (data not shown). Purification can be achieved by
classical chromatographic techniques, as described in
Experimental. Such ionized aromatics including hydroxyl
groups seem to be very important as starting or condensing
materials for the chemical synthesis of biologically active
organic molecules such as therapeutic agents.
Figure 2. Application of modified Mosher’s method in determination of the
absolute configuration of 1-[((5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl)-
methyl]piperidin-4-one (8).
the product (9) was determined to be C12H19NO4 by
HR-EIMS. Analysis by HMQC and DQF COSY spectra
showed that 9 was dihydrodiol derivative. The 2,3-diol
regiochemical assignment was confirmed by the long range
1H–13C connectivities from H-10 (d 3.75) to C-1 (d 138.1),
C-2 (d 68.6) and C-6 (d 120.4). Thus, 9 was determined to
be (5,6-dihydroxy-cyclohexa-1,2-dienylmethyl)-carbamic
acid tert-butyl ester (Fig. 1).
In conclusion, the present study and our previous report3
have shown that the use of evolved biphenyl dioxygenases is
effective for the synthesis of high value organic molecules
used in the industrial sector. Many of the products generated
by this study are difficult to be synthesized with existing
methods of organic chemistry. It would be feasible to
complement the methods of combinatorial chemistry with
the biotechnological methods described in our papers. We
aim to establish such biological technologies alongside
combinatorial chemistry, in an approach we term ‘Bio-
CombiChem’ (Biology-based Combinatorial Chemistry).
2.3.2. Phenyl-carbamic acid tert-butyl ester. Phenyl-
carbamic acid tert-butyl ester was converted to one product
by the S. lividans transformant. The molecular formula of
the product (10) was determined to be C11H15NO3 by HR-
EIMS. Analysis by HMQC and DQF COSY spectra
revealed that one phenolic OH was attached at benzene
ring. C-4 (d 68.2). Observation of visinal sp2 spin networks
from H-3 (d 6.89) to H-6 (d 7.03) confirmed that the OH
function was attached at C-2. Thus, 10 was determined to be
(2-hydroxy-phenyl)-carbamic acid tert-butyl ester (Fig. 1).
4. Experimental
4.1. Plasmids, bacterial strains, and growth conditions
3. Discussion
Plasmid pKF2072 carrying the bphA1(2072)A2A3A4
genes for their expression in E. coli has been described.3
E. coli JM10912 was used as a host for plasmid pKF2072,
and cultured in LB medium12 or M9 medium12 at 30 or
378C. Ampicillin (Ap) (50–150 mg/ml) was added when
needed.
Traditionally, biphenyl dioxygenase catalyzes the formation
of cis-1,2-dihydrodiol from a benzene ring.1 In this sense,
compounds 8 and 9 were typical products converted by this
enzyme. However, the other products formed were not cis-
1,2-dihydrodiol, but monohydroxylated or trihydroxylated
products. The monohydoxylated forms are considered to be
generated non-enzymatically by dehydration from the
corresponding 1,2-dihydrodiols, as a consequence of their
structural instability. The reason is not clear why in the case
of product 7 a catechol was generated instead of cis-
dihydrodiol from a benzene ring. But, the similar catechol-
type products have also shown to be produced by the
modified biphenyl dioxygenase-mediated transformation of
flavone and flavanone.4 Endogenous desaturation enzymes,
which are responsible for the conversion from cis-
dihydrodiol to catechol, may exist in the Streptomyces
species used.
The expression vector pIJ6021 for Streptomyces species,
which carries the kanamycin resistance gene and thio-
strepton-inducible promoter PtipA has been described.13
A
plasmid, pIJ6021-bphA1(2072)A2A3A4, for the expression
of the modified biphenyl dioxygenase genes in S. lividans
has been constructed using vector pIJ6021.4 On plasmid
pIJ6021-bphA1(2072)A2A3A4, the four genes are
positioned under the control of the thiostrepton-inducible
promoter PtipA enabling co-transcription in S. lividans.
S. lividans TK2114 was used as a host for this plasmid,
and cultured in YEME medium15 or minimal medium15
at 308C. Kanamycin was used at a final concentration of
5 mg/ml when necessary.
This study has shown an advantage of using Streptomyces
species instead of E. coli as hosts for bioconversion
experiments. It is significant that the E. coli transformant
expressing the modified dioxygenase genes was found not to
convert 1-naphthoic acid and 2-(1-naphthyl)acetic acid,
which were converted by the Streptomyces transformant. It
is possible that the difference would depend principally on
better permeability of ionized substrates through the cell
membrane in Streptomyces species (gram positive bacteria)
compared with E. coli (gram negative bacterium).
4.2. Conversion experiments
E. coli JM109 harboring pKF2072 was grown in LB
medium containing 150 mg/ml of Ap at 308C with
reciprocal shaking (175 rpm) for 8 h. Five milliliters of
this culture was inoculated into 100 ml of M9 medium with
150 mg/ml of Ap, 10 mg/ml of thiamine, and 0.4% (w/v)
glucose in Erlenmeyer flask at 308C with reciprocal shaking
(175 rpm) for 16–17 h, of which the absorbance in OD

K. Shindo et al. / Tetrahedron 59 (2003) 1895–1900
1899
600 nm reaches approximately 1. One millimolar (the final
concentration) of isopropyl b-D-thiogalactopyranoside
(IPTG) was added to the culture, and further cultivated for
4 h. The cells were collected by centrifugation, washed once
with M9 medium, and then resuspended in 100 ml of fresh
M9 medium with 150 mg/ml of Ap, 10 mg/ml of thiamine,
0.4% (w/v) glucose, and 1 mM (the final concentration) of
IPTG, along with 100 mg/ml (the final concentration) of
each substrate, and cultivated in Erlenmeyer flask at 308C
with reciprocal shaking (175 rpm) for 2–3 days.
157.8 (C-4), 168.2 (C-10). (Found: Mþ, 188.0470. Calcd for,
188.0473 (C11H8O3)).
Compound 2. 1H NMR (DMSO-d6): 6.92 (d, 1H, J¼7.3 Hz),
7.40 (dd, 1H, J¼7.3, 7.3 Hz), 7.48 (dd, 1H, J¼7.3, 7.3 Hz),
8.07 (dd, 1H, J¼2.0, 7.3 Hz), 8.24 (d, 1H, J¼7.3 Hz), 8.37
(dd, 1H, J¼2.0, 7.3 Hz); 13C NMR (DMSO-d6) d: 108.2
(C-6), 116.2 (C-8), 123.5 (C-3), 125.1 (C-4a), 126.7 (C-4),
127.7 (C-1), 128.10(C-7), 130.0 (C-2), 132.0 (C-8a), 153.6
(C-5), 168.8 (C-1 ). (Found: Mþ, 188.0466. Calcd for,
188.0473 (C11H8O3)).
S. lividans TK21 harboring pIJ6021-bphA1(2072)A2A3A4
was grown in 100 ml of YEME medium containing 5 mg/ml
of kanamycin in a shaking (Sakaguchi) flask at 308C with
reciprocal shaking (120 rpm) for 2 days. One milliliter of
this culture was inoculated into the same medium, and
cultivated under the same conditions. After 24 h, thio-
streptone was added to the culture at a final concentration of
5 mg/ml to induce transcription from PtipA. After an
additional 24 h of incubation the mycelium was collected
by centrifugation and washed once with minimal medium.
Then 100 mg (wet weight) mycelium was resuspended in
100 ml of fresh minimal medium, and 10 mg or 1 mM (the
final concentration) of each substrate was added to the
mycelium suspension. The mycelium and substrates were
incubated on a reciprocal shaker (120 rpm) at 308C for 24 h,
followed by HPLC analysis of the culture supernatant. All
the substrates were purchased from Aldrich or Sigma.
4.3.2. 2-(4-Hydroxyl-1-naphthyl)acetic acid (3) and 2-(5-
hydroxyl-1-naphthyl)acetic acid (4) (product converted
from 2-(1-naphthyl)acetic acid). The crude EtOAc extract
(80 mg) was subjected to column chromatography
(CH2Cl2–MeOH¼15:1) to yield 24.6 mg of 3 and 6.9 mg
of 4.
Compound 3. 1H NMR (CDCl3) d: 3.96 (s, 2H), 6.72 (d, 1H,
J¼7.9 Hz), 7.18 (d, 1H, J¼7.9 Hz), 7.45 (dd, 1H, J¼7.9,
7.9 Hz), 7.51 (dd, 1H, J¼7.9, 8.5 Hz), 7.88 (d, 1H,
J¼8.5 Hz), 8.22 (d, 1H, J¼7.9 Hz); 13C NMR (CDCl3) d:
38.0 (C-10), 113.5 (C-3), 122.0 (C-5), 122.1 (C-1), 123.7
(C-8), 125.4 (C-7), 125.7 (C-4a), 126.5 (C-6), 128.2 (C-2),
133.7 (C-8a), 152.3 (C-4), 175.5 (C-20) (Found: Mþ
202.0633. Calcd for 202.0630 (C12H10O3)).
Compound 4. 1H NMR (CDCl3) d: 4.03 (s, 2H), 6.80 (d, 1H,
J¼7.3 Hz), 7.30 (dd, 1H, J¼7.3, 7.3 Hz), 7.39 (2H), 7.51 (d,
1H, J¼7.3 Hz), 8.16 (dd, 1H, J¼2.5, 7.9 Hz); 13C NMR
(CDCl3) d: 39.2 (C-10), 108.5 (C-6), 116.0 (C-8), 122.3
(C-4), 124.5 (C-3), 124.9 (C-4a), 126.6 (C-7), 128.5 (C-2),
130.2 (C-1), 133.4 (C-8a), 153.0 (C-5), 174.1 (C-20).
(Found: Mþ202.0631. Calcd for 202.0630 (C12H10O3)).
4.3. Purification and identification of converted products
The culture supernatant (about 1 l) was extracted with 1 l of
ethyl acetate (EtOAc). The organic layer was concentrated
in vacuo, and analyzed by thin-layer chromatography (TLC)
on silica gel (0.25 mm Silica Gel 60 (Merck)). The solvent
systems were as follows: 1-naphthoic acid, CH2Cl2–MeOH
(10:1); 2-(1-naphthyl)acetic acid, CH2Cl2–MeOH (10:1);
diphenylamine, CH2Cl2; 1-benzyl-4-piperidone, CH2Cl2–
MeOH (10:1); benzyl-carbamic acid tert-butyl ester,
CH2Cl2–MeOH (10:1); phenyl-carbamic acid tert-butyl
ester, hexane–EtOAc (3:1). The converted products as well
as the substrates, present in the organic phase, were put
through column chromatography on silica gel (20 by
250 mm, Silica Gel 60 (Merck)).
4.3.3. 2-Anilinophenol (5), 4-anilinophenol (6), and 4-(2-
hydroxyanilino)-1,2-benzenediol (7) (products converted
from diphenylamine). The crude EtOAc extract (234 mg)
was subjected to column chromatography (Hexane–
CH2Cl2¼1:1 to CH2Cl2–MeOH¼100:1, stepwise) to yield
46.3 mg of 5, 8.4 mg of 6 and 3.4 mg of 7.
1
Compound 5. H NMR (CDCl3) d: 5.14 (drs, 1H), 5.72 (s,
1H), 6.68 (d, 2H, J¼7.9 Hz), 6.79 (dd, 1H, J¼7.9, 7.9 Hz),
6.80 (dd, 1H, J¼7.9, 7.9 Hz), 6.88 (d, 1H, J¼7.9 Hz), 6.99
(dd, 1H, J¼7.9, 7.9 Hz), 7.08 (d, 1H, J¼7.9 Hz), 7.13 (dd,
2H, J¼7.9, 7.9 Hz); 13C NMR (CDCl3) d: 115.3 (C-3),
115.8 (C-20, C-60), 120.3 (C-40), 121.0 (C-5), 124.5 (C-6),
126.0 (C-4), 129.4 (C-30, C-50), 129.1 (C-1), 145.3 (C-10),
150.9 (C-2). (Found: Mþ 185.0845. Calcd for 185.0841
(C12H11NO)).
The structures of the converted products were analyzed by
mass (MS) (EI-MS, JEOL DX-303) and nuclear magnetic
resonance (NMR) (500 MHz, JEOL a) spectra. TMS was
used for the internal standard. (R)- and (S)-2NMA esters
were prepared in a manner reported by Kusumi et al.16
4.3.1. 4-Hydroxy-1-naphthoic acid (1) and 5-hydroxy-1-
naphthoic acid (2) (product converted from 1-naphthoic
acid). The crude EtOAc extract (90 mg) was subjected to
column chromatography (CH2Cl2–MeOH¼15:1) to yield
15.6 mg of 1 and 24.0 mg of 2.
Compound 6. 1H NMR (CDCl3) d: 4.64 (brs, 1H), 5.42 (brs,
1H), 6.76 (d, 2H, J¼8.6 Hz), 6.80 (dd, 1H, J¼7.3, 7.3 Hz),
6.87 (d, 2H, J¼7.3 Hz), 6.99 (d, 2H, J¼8.6 Hz), 7.18 (dd,
2H,0 J¼7.3, 7.3 Hz); 13C NMR (CDCl3) d: 115.7 (C-20,
C-6 ), 116.1 (C-3, C-5), 119.6 (C-40), 122.4 (C-2, C-6),
129.3 (C-30, C-40), 135.8 (C-1), 145.1 (C-10), 151.1 (C-4).
(Found: Mþ 185.0847. Calcd for 185.0841 (C12H11NO)).
Compound 1. 1H NMR (DMSO-d6) d: 6.90 (d, 1H,
J¼7.9 Hz), 7.49 (dd, 1H, J¼7.3, 7.3 Hz), 7.58 (dd, 1H,
J¼7.3, 9.1 Hz), 8.12 (d, 1H, J¼7.9 Hz), 8.22 (d, 1H,
J¼7.3 Hz), 9.02 (s, 1H, J¼9.1 Hz); 13C NMR (DMSO-d6)
d: 106.9 (C-3), 117.0 (C-1), 124.6 (C-4a), 124.7 (C-6), 125.5
(C-8), 127.8 (C-7), 132.8 (C-5), 132.8 (C-2), 132.9 (C-8a),
Compound 7. 1H NMR (CDCl3) d: 6.31 (d, 1H, J¼1.8 Hz),
6.84 (dd, 1H, J¼1.8, 9.8 Hz), 7.32 (d, 1H, J¼8.6 Hz), 7.35

1900
K. Shindo et al. / Tetrahedron 59 (2003) 1895–1900
(dd, 1H, J¼7.9, 8.6 Hz), 7.42 (d, 1H, J¼9.8 Hz), 7.52 (ddd,
1H, J¼1.2, 8.6, 8.6 Hz), 7.77 (dd, 1H, J¼1.2, 7.9 Hz); 130C
NMR (CDCl3) d: 107.0 (C-2), 116.2 (C-30), 125.5 (C-5 ),
130.5 (C-60), 132.9 (C-40), 133.4 (C-10), 134.8 (C-5), 135.3
(C-6), 144.1 (C-2), 148.5 (C-4), 149.6 (C-3), 155.0 (C-1).
(Found: Mþ 217.0733. Calcd for 217.0739 (C12H11O3)).
Acknowledgements
We wish to thank Dr Tadashi Eguchi and Dr Yoshitaka
Matsushima (Tokyo Institute of Technology) for valuable
advice. We also wish to thank Dr Paul D. Fraser (Royal
Holloway University of London) for careful reading of the
manuscript.
4.3.4. 1-[(5,6-Dihydroxycyclohexa-1,3-dienyl)methyl]-
piperidin-4-one (8) (product converted from 1-benzyl-
4-piperidone). The crude EtOAc extract (536 mg) was
subjected to column chromatography (CH2Cl2–
MeOH¼30:1) to yield 68.0 mg of 8.
References
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1
Compound 8. H NMR (CDCl3) d: 2.36 (dd, 2H, J¼6.1,
6.1 Hz), 2.64 (ddd, 1H, J¼6.1, 12.2, 12.2 Hz), 2.81 (ddd,
1H, J¼6.1, 12.2, 12.2 Hz), 3.05 (d, 1H, J¼12.8 Hz), 3.40 (d,
1H, J¼12.8 Hz), 4.02 (m, 1H), 4.38 (d, 1H, J¼5.5 H0z), 5.70
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52.4 (C-30, C-70), 61.0 (C-10), 65.9 (C-3), 72.0 (C-2), 1220.2
(C-6), 125.3 (C-4), 127.8 (C-5), 135.4 (C-1), 207.7 (C-5 ).
(Found: Mþ 223.1212. Calcd for 223.1209 (C12H17NO3)).
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Compound 9. H NMR (CDCl3) d: 1.37 (s, 9H), 3.75 (dd,
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(C-2), 79.9 (C-50), 120.4 (C-6), 124.4 (C-5), 128.7 (C-4),
138.1 (C-1), 156.4 (C-30). (Found: Mþ 241.1312. Calcd for
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1H), 6.78 (ddd, 1H, J¼1,2, 7.3, 7.9 Hz), 6.89 (dd, 1H,
J¼1.2, 7.8 Hz), 6.95 (ddd, 1H, J¼1.7, 7.3, 7.8 Hz), 7.03 (dd,
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121.4 (C-6), 125.6 (C-1), 125.6 (C-4), 147.4 (C-2), 155.0
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(C11H15NO3)).
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