Biosynthesis of tetrapetalones

Article abstract of DOI:10.1039/b618425a

The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was studied by feeding experiments with 1- ¹³C sodium propanoate, 1-¹³C sodium butanoate, carbonyl-¹³C 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and 1-¹³C glucose, followed by analysis of the ¹³C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1) is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the biosynthetic relationship of the four tetrapetalones was indicated. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b618425a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Biosynthesis of tetrapetalones
Toshikazu Komoda,*a,b Yasumasa Sugiyamaa and Akira Hirotaa
Received 18th December 2006, Accepted 23rd March 2007
First published as an Advance Article on the web 17th April 2007
DOI: 10.1039/b618425a
The biosynthesis of tetrapetalones (tetrapetalones A, B, C, and D) in Streptomyces sp. USF-4727 was
studied by feeding experiments with [1-13C] sodium propanoate, [1-13C] sodium butanoate,
[carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA) hydrochloride, and [1-13C] glucose, followed by
analysis of the 13C-NMR spectra. These feeding experiments revealed that the four tetrapetalones were
polyketide compounds constructed from propanoate, butanoate, AHBA, and glucose. The
tetrapetalone biosynthetic pathway was also suggested in this study. In this pathway, tetrapetalone A (1)
is synthesized by polyketide synthase (PKS) using AHBA as a starter unit, then the side chain of 1 is
subjected to acetoxylation to produce tetrapetalone B (2). Additionally, 1 is oxidized and transformed
into tetrapetalone C (3). In a similar way, 2 is converted to tetrapetalone D (4). Therefore, the
biosynthetic relationship of the four tetrapetalones was indicated.
was novel, we investigated the biosynthetic precursors and the
biosynthetic pathway to these tetrapetalones.
Introduction
We are investigating the discovery of new bioactive compounds
from microorganisms by using a lipoxygenase assay.1 Human
lipoxygenase and cyclooxygenase catalyze the first step in the
arachidonic pathway, resulting in the production of some impor-
tant signaling molecules that may be involved in a variety of human
diseases.2–4 Soybean lipoxygenase is used for our screening assay.
This enzyme shows 15-lipoxygenase activity in arachinonate.
Tetrapetalones A (1), B (2), C (3), and D (4), were isolated
from the culture broth of Streptomyces sp. USF-4727 as inhibitors
of lipoxygenase.5–7 Their chemical structures were elucidated by
using spectroscopic methods to show that these compounds are
constructed with a characteristic tetracyclic skeleton and a sugar
moiety. Because the tetracyclic skeleton of these tetrapetalones
Results and discussion
To explore the biosynthetic origins of tetrapetalones, we added
13C-labeled compounds to the culture of the tetrapetalone-
producing strain, Streptomyces sp. USF-4727. After isolation of
each tetrapetalone from this culture, the incorporation ratio of the
13C-labeled compounds was evaluated.
In our feeding experiment of 13C-labeled compounds, we
identified that [1-13C] propanoate, [1-13C] butanoate, [1-13C] glu-
cose and [carbonyl-13C] AHBA are efficiently incorporated into
tetrapetalone A (1), and that the incorporation rate of [1-13C] and
[2-13C] acetate into 1 was relatively low level in this experiment
(Table 1).
As shown in Table 1, the peaks of C-1, -5, and -7 were highly
enriched (each enrichment ratio >30.0) in the experiments with
[1-13C] propanoate. The carbons C-3 and C-9 were also remarkably
enriched (each enrichment ratio >45.0) in the experiments with
[1-13C] butanoate and [carbonyl-13C] AHBA, respectively.
These results indicated that the aglycon of 1 is constructed from
three molecules of propanoate, one butanoate, and one AHBA, as
shown in Fig. 1.
aLaboratory of Applied Microbiology, School of Food and Nutritional
Sciences, University of Shizuoka, Suruga-ku Yada 52-1, Shizuoka, Japan
422-8526. E-mail: komoda@myu.ac.jp; Fax: +81-22-245-1534
bSchool of Food, Agricultural and Environmental Sciences, Miyagi Univer-
sity, Taihaku-ku Hatatate 2-2-1, Sendai, Japan 982-0215
Fig. 1 Biosynthetic precursors for tetrapetalone A.
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Table 1 Incorporation of 13C-labeled compoundsa
Enrichment ratio (tetrapetalone A)
Enrichment ratio (tetrapetalone B)
[1-13C]AA
Position
[1-13C]AA
[2-13C]AA
[1-13C]PA
[1-13C]BA
[1-13C]G
13C-AHBA
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
3.4b
1.4
3.2
0.6
3.8
1.3
3.5
1.3
1.2
1.2
0.9
0.8
0.8
0.7
0.6
1.1
6.3
0.7
1.1
1.1
0.9
0.9
1.5
1.3
1.8
1.0
—
3.0
0.9
1.5
3.1
2.5
2.5
2.9
3.6
0.8
0.6
1.0
0.6
0.8
0.6
0.9
0.6
0.9
3.0
3.3
3.2
1.5
1.0
1.2
1.0
1.4
1.0
—
39.0
1.1
0.9
0.5
37.5
0.3
34.4
0.5
0.9
1.1
1.1
1.1
1.1
0.9
0.4
1.0
1.1
0.8
0.7
1.2
0.9
1.0
1.2
1.2
1.2
1.0
—
3.2
0.7
45.1
0.5
5.4
0.6
0.6
0.7
0.8
1.1
1.0
0.5
0.7
0.6
0.7
0.7
1.0
0.7
0.9
1.0
0.8
0.9
1.4
1.2
1.4
1.0
—
Ndc
1.7
1.3
1.1
1.3
1.2
1.0
1.1
0.7
0.9
1.5
0.6
0.8
0.7
1.3
1.7
0.9
0.9
1.3
1.3
2.1
0.9
1.2
0.9
1.3
1.0
—
1.5
1.4
1.7
1.0
1.5
0.7
1.0
0.5
58.3
0.6
1.2
0.7
1.3
0.8
0.7
1.5
1.5
0.9
1.2
1.2
1.0
0.9
1.4
1.2
1.5
1.0
—
1.0
0.7
1.5
1.0
1.7
1.3
2.1
1.4
1.3
0.9
0.6
1.0
1.0
1.0
1.0
0.9
2.8
0.9
0.8
0.7
0.7
0.9
1.1
1.0
1.2
1.0
4.4
0.8
17
18
19
20
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
6ꢀ
17-OCOCH3
17-OCOCH3
—
—
—
—
—
—
a [1-13C]AA: CH313COONa, [2-13C]AA: 13CH3COONa, [1-13C]PA: CH3CH213COONa, [1-13C]BA: CH3CH2CH213COONa, [1-13C]G: [1-13C] glucose, 13C-
AHBA: [carbonyl-13C] 3-amino-5-hydroxybenzoic acid. b Enrichment ratio was calculated from the relative intensity of C-6ꢀ as 1.0. c Not detected.
However, the relatively low incorporation rate of [1-13C] and
[2-13C] acetate into 1 suggested that acetate was not directly
incorporated into 1. In fatty acid biosynthesis and in macrolide
antibiotic biosynthesis, two molecules of acetyl-CoA could be
joined in a Claisen condensation to form an acetoacetyl-CoA.8,9
This condensation might be observed in our 13C-labeled sodium
acetate feeding experiment to produce 1. The four carbons C-
3, -4, -17, and -18 were shown to be derived from butanoate
described above. However, C-3 and C-17 were also enriched in
the [1-13C] sodium acetate feeding experiment. The [2-13C] sodium
acetate feeding experiment lead to the enhancement of the signal
intensity at C-4 and C-18 in the 13C-NMR spectrum, showing
that this C4 unit was formed with two molecules of acetate in
a head-to-tail pattern. Other relatively low enrichment ratios in
the 13C-labeled acetate feeding experiment also suggested that
acetate was used in forming the C3 unit (i.e. methylmalonyl-
CoA) derived from propanoate, then incorporated into tetrapeta-
lone A.
In the [1-13C] glucose feeding experiment, the enrichment ratio
of C-1ꢀ was 2.1, indicating that the 2,3,6-trideoxy-D-galactosyl
moiety of 1 is derived from glucose. This relatively low enrichment
ratio (2.1) was explained as follows: because glucose is a major
carbon source for microorganisms, [1-13C] glucose fed to the
culture broth of USF-4727 is not consumed in the tetrapetalone
biosynthetic system alone, but is also distributed to many other
metabolic and biosynthetic systems. Therefore, only a part of the
labeled glucose was incorporated into 1 to give the relatively low
enrichment ratio at the C-1ꢀ position in the 13C-NMR spectrum.
Similar low enrichment ratios are often observed in other 13C-
labeled glucose feeding experiments.10–12
The origin of the carbon atoms in tetrapetalone B (2), except
for the acetoxy group joining to the carbon at C-17, was estimated
by the result of the feeding experiments for tetrapetalone A (1)
described above. In this study, it was also revealed that the acetoxy
group in 2 is derived from an acetate unit.
As shown in Table 1, the carbonyl carbon of the acetoxy
group joined to C-17 in 2 was enriched (enrichment ratio: 4.4)
by feeding with [1-13C] sodium acetate. This result suggests that
the labeled acetate was used for constructing the C-17 side chain
of 2. Moreover, [1,2-13C2] the sodium acetate feeding experiment
supported this suggestion. In the feeding experiment with [1,2-
13C2] sodium acetate, we observed a JC–C coupling (coupling
constant: 59 Hz) only between the carbonyl carbon and the acetoxy
methyl carbon in the 13C-NMR spectrum of 2. This observation
indicated that these two carbons are constructed with the [1,2-
13C2] acetate and that the side chain at C-17 in 2 is derived from
an acetate unit.
Subsequently, we proposed a transformational scheme for
tetrapetalones (A, B, C and D) by the results of these feed-
ing experiments and the oxidation experiment of 1 described
below.
To explore the chemical properties of 1, we treated 1 with H2O2
(aq.) in 0.2 M borate buffer (pH 9.0). The reaction mixture was
analyzed by HPLC every 30 minutes. In the reaction mixture
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of 1 with H2O2 (aq.), the concentration of 1 was gradually
decreased, while an unknown compound emerged in the HPLC
chromatogram as shown in Fig. 2. In the control test, using
H2O instead of H2O2, no emergence of the unknown compound
was observed. This observation showed that 1 is transformed to
another compound by treatment with H2O2. We also observed a
moderate reduction in the concentration of 1 in the control test
of this experiment, showing that 1 is not completely stable under
these conditions.
chromophores in several ansamycin antibiotics.13,14 In this report,
it is revealed that the tetrapetalones are polyketide compounds
constructed by AHBA, propanoate, butanoate, and glucose, indi-
cating that tetrapetalones are biologically synthesized in a similar
scheme to the ansamycin antibiotics. The ansamycin antibiotics
have a common structural feature which is the “ansa” bridge
system of polyketide origin initiated from AHBA as a starter unit.
In the tetrapetalone biosynthetic pathway, a compound which
has an “ansa” bridge system in its chemical structure should be
synthesized as an intermediate compound, and then this bridge
should be modified to form the tetracyclic structure. The details
of the formation process of the tetracyclic skeleton are now under
investigation.
The transformational scheme of tetrapetalones proposed due
to this study is shown in Fig. 3. At first, tetrapetalone A (1) is
synthesized by polyketide synthase using AHBA as a starter unit.
After receiving an oxygen atom to give the hydroxy group at C-17
in 1, the side chain of 1 should be subjected to acetoxylation,
then form tetrapetalone B (2). On the other hand, 1 and 2
should be oxidized and transformed to tetrapetalones C (3)
and D (4), respectively, in the culture broth of Streptomyces sp.
USF-4727.
In this study, we were able to reveal the origins of tetrapetalones
A, B, C, and D and indicate their proposed transformational
scheme.
Experimental
Chemicals
[1-13C] and [2-13C] sodium acetate, [1-13C] sodium butanoate,
and [1-13C] glucose were purchased from Sigma Co. (St. Louis,
MO, USA). [1-13C] sodium propanoate, and [carbonyl-13C] ben-
zoic acid were obtained commercially from Cambridge Isotope
Laboratories, Inc. (Andover, MA, USA). Other chemicals were
purchased from Wako Pure Chemical Industries, Ltd. (Osaka,
Japan). [Carbonyl-13C] 3-amino-5-hydroxybenzoic acid (AHBA)
hydrochloride was prepared in our laboratory as described
below.
Fig. 2 Reaction of tetrapetalone A with H2O2: (a) treatment with
H2O2 (aq.), (b) treatment with H2O (control), ᭺: tetrapetalone A, ᭹:
tetrapetalone C.
Instruments
Spectroscopic measurements were taken with the following instru-
ments: NMR, JEOL Alpha-400 spectrometer (tetramethylsilane
as internal reference at 0 ppm for 1H- and 13C-NMR); FAB-MS,
JEOL JMS-700 spectrometer; UV and visible spectra, Shimadzu
UV-160A spectrometer; melting point, Yanagimoto MP-J3; IR,
Jasco FT/IR-550; HPLC for analysis, Jasco PU-2080 Plus, Jasco
UV 2077 Plus, Jasco MX-2080-32, and HPLC for preparation;
Jasco 880-PU, Jasco UV-970.
For the structure elucidation of the unknown compound, we
treated 65 mg of 1 with H2O2 (aq.). After purification of the
reaction mixture, we obtained 30 mg of the reaction product. The
1H- and 13C-NMR spectral data, HPLC retention time and the
optical rotation [a]D of this product were completely consistent
with those of tetrapetalone C (3), thereby identifying this reaction
product as 3. These results indicated that 1 was subject to oxidation
under these in vitro conditions, and was then transformed to 3.
In a similar way, 2 is implied to be converted to tetrapetalone
D (4), indicating that 1 and 2 might be converted to 3 and 4,
respectively, in the culture broth of the tetrapetalone-producing
strain.
Cultivation of a Streptomyces sp USF-4727 strain
The strain USF-4727 was inoculated into 800 mL of the medium
(0.4% glucose, 0.4% yeast extract, 1.0% malt extract, pH 7.3) in a
2 litre Erlenmeyer flask and cultivated at 30 ◦C for 10–12 days on
a rotary shaker (130 rpm).
The 3-amino-5-hydroxybenzoic acid (AHBA) unit is suggested
as the biological starter unit for the meta-C7N units of the aromatic
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Fig. 3 Proposed transformational scheme for tetrapetalones.
Isolation of tetrapetalones A (1), B (2), C (3), and D (4)
(e 10350), 250 nm (sh. e 5890), 311 nm (e 3190), 350 nm (sh. e
2900). IR mmax (KBr) cm−1: 3400, 1720, 1630, 1380, 1280, 1170,
1060, 1020. The 1H- and 13C-NMR data in CD3OD are shown in
our previous paper.6
The culture filtrate of Streptomyces sp. USF-4727 strain was
purified by chromatography on a Diaion HP-20 column, silica
gel column, Sephadex LH-20 (MeOH), and preparative HPLC
to yield 1, 2, 3, and 4. The details of the isolation of the four
tetrapetalones are described in our previous papers.5,6
Tetrapetalone D (4). Colorless amorphous powder; melting
◦
point, 148–151 C. HRFAB MS [M + H]+, m/z 546.2352
(546.2339 calcd. for C28H36NO10). UV–VIS kmax (MeOH): 208 nm
(sh. e 9920), 247 nm (sh. e 4470), 312 nm (e 1720), 335 nm (sh.
e 1440). IR mmax (KBr) cm−1: 3420, 1720, 1630, 1380, 1240, 1160,
Tetrapetalone A (1). Pale yellow amorphous powder; melting
◦
point, 190 C. HRFAB MS [M + H]+, m/z 472.2354 (472.2335
calcd. for C26H34NO7). UV–VIS kmax (MeOH): 219 nm (e 18300),
245 nm (sh. e 13300), 340 nm (e 11700). IR kmax (KBr) cm−1: 3400,
1670, 1380, 1300, 1250, 1170, 1060, 1020. The 1H- and 13C-NMR
data in CD3OD are shown in our previous paper.5
1
1120, 1060, 1020. The H- and 13C-NMR data in CD3OD are
shown in our previous paper.6
Feeding experiments with [1-13C] and [2-13C] sodium acetate, and
[1-13C]sodium propanoate
Tetrapetalone B (2). Pale yellow amorphous powder; melting
◦
point, 191–193 C. HRFAB MS [M + H]+, m/z 530.2407
(530.2390 calcd. for C28H36NO9). UV–VIS kmax (MeOH): 217 nm (e
9310), 336 nm (e 4190). IR mmax (KBr) cm−1: 3420, 1580, 1570, 1560,
1540, 1250. The 1H- and 13C-NMR data in CD3OD are shown in
our previous paper.6
[1-13C] Sodium acetate (800 mg), [2-13C] sodium acetate (800 mg),
and [1-13C] sodium propanoate (400 mg) were added to the culture
of Streptomyces sp. USF-4727 strain in 800 mL of medium (0.4%
glucose, 0.4% yeast extract, 1.0% malt extract, pH 7.3) on day 5–7
from the beginning of the incubation. At day 10–12, each culture
was filtered and the tetrapetalones in these cultures were isolated
and submitted to further investigation.
Tetrapetalone C (3). Colorless amorphous powder; melting
◦
point, 154–157 C. HRFAB MS [M + H]+, m/z 488.2286
(488.2284 calcd. for C26H34NO8). UV–VIS kmax (MeOH): 206 nm
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Feeding experiment with [1,2-13C2] sodium acetate
labeled AHBA. However, in the 13C-NMR spectrum (CD3OD,
100 MHz) of [carbonyl-13C] AHBA, the carbon signal at 168.0 ppm
was extremely enriched, at least 100 times higher than that of non-
labeled 3-amino-5-hydroxybenzoic acid.
[1,2-13C2] Sodium acetate was added to two of the cultivative
Erlenmeyer flasks (each 300 mg) containing 800 mL of culture
at day 7 with non-labeled sodium propanoate (800 mg) and non-
labeled sodium butanoate (250 mg). On day 10, the culture broth
was filtered and each tetrapetalone was isolated and submitted to
further investigation.
Feeding experiment with [carbonyl-13C] 3-amino-5-hydroxybenzoic
acid (AHBA) hydrochloride
[Carbonyl-13C] AHBA hydrochloride (200 mg) was added to the
culture with non-labeled sodium propanoate (800 mg) and non-
labeled sodium butanoate (250 mg) on day 7 from the beginning
of incubation. On day 10, the culture was filtered, and each
tetrapetalone in the culture was isolated and used for further
investigation.
Feeding experiment with [1-13C] sodium butanoate
[1-13C] Sodium butanoate (250 mg) was added to the culture
(800 mL) with non-labeled sodium propanoate (800 mg) on day
7 from the beginning of incubation. On day 10, the culture was
filtered, and each tetrapetalone in the culture was isolated and
used for further investigation.
Evaluation of the incorporation of 13C-labeled precursors
We evaluated the incorporation ratio of each 13C-labeled com-
pound by measurement of the 13C-NMR spectrum. Tetrapetalones
A (1) and B (2) isolated from the each culture broth to which were
added the 13C-labeled compounds were subject to measurement of
the 13C-NMR spectrum. The signal intensity of each peak in this
spectrum was measured to evaluate the signal enrichment. The
enrichment ratio was given from each peak height compared to
the standard peak height (C-6ꢀ) in each 13C-NMR spectrum.
Feeding experiment with [1-13C] glucose
The culture medium of this feeding experiment was different from
other feeding experiments. The strain USF-4727 was inoculated to
the “glucose-less” medium (0.4% yeast extract, 1.0% malt extract,
pH 7.3). The cultivation was done in a similar way to other feeding
experiments. On day 5 after the inoculation of the tetrapetalone-
producing strain, [1-13C] glucose (100 mg) was added to the culture
with non-labeled sodium propanoate (800 mg). On day 10, the
culture was filtered, and each tetrapetalone in the culture was
isolated in a similar way to the other feeding experiments.
Treatment of tetrapetalone A (1) with H2O2
First, we treated 1 with H2O2 solution for HPLC analysis. Two
hundred microlitres of 30% H2O2 (aq.) were added to 0.2 mg
of 1 in 0.5 mL of 0.2 M borate buffer (pH 9.0) to start the
reaction. The mixture was analyzed by HPLC (25% CH3CN–
10 mM phosphate buffer (pH 2.6), Capcell Pak C18 SG120, φ
4.6 × 250 mm, UV 254 nm) every 30 minutes. Then we treated
1 with H2O2 for the HPLC preparation. Six millilitres of 30%
H2O2 (aq.) were added to the 65 mg of tetrapetalone A in 10 mL
of 0.2 M borate buffer (pH 9.0), standing for 180 min. The
mixture was applied to the Diaion HP-20 (100 g) column. After
being washed with water, the reaction product was eluted with
MeOH. The MeOH fraction was purified by preparative HPLC
(25% CH3CN–10 mM phosphate buffer (pH 2.6), Capcell Pak C18
SG120, φ 15 × 250 mm, UV 254 nm) to yield 30 mg of the reaction
product. The chemical structure of this compound was elucidated
by spectroscopic methods.
Preparation of [carbonyl-13C] 3-amino-5-hydoxybenzoic acid
(AHBA) hydrochloride
First, we prepared non-labeled AHBA hydrochloride on the basis
of the method by Becker et al.15 After structure elucidation of this
non-labeled compound by spectroscopic methods, we referred to
the spectral data for the structure elucidation of [carbonyl-13C]
AHBA hydrochloride. In the preparation of non-labeled AHBA
hydrochloride, benzoic acid was used as the starting compound.
First, benzoic acid was treated with fuming sulfuric acid to form
3,5-disulfonicbenzoic acid. The reactant was neutralized with
barium carbonate. This barium salt was reacted with sodium and
potassium hydroxides. After dissolving in water, the reactant was
extracted with diethyl ether (pH 3.0) to give 3,5-dihydroxybenzoic
acid.16 Then, this 3,5-dihydroxybenzoic acid was converted to
AHBA hydrochloride by reaction with NH4Cl and 28% aq. NH3
in a steel bomb at 180 ◦C for 40 h.8 Finally, we obtained AHBA
hydrochloride (1.8 g, 58%) by using benzoic acid (2.0 g) as a
starting compound. 1H-NMR of non-labeled AHBA (400 MHz;
CD3OD; Me4Si) dH: 7.05 (1 H, t, J 2.3 Hz), 7.49 (1 H, m), 7.52
(1 H, m); 13C-NMR of 3-amino-5-hydroxybenzoic acid (100 MHz;
CD3OD) dC: 115.4 (d), 115.7 (d), 118.0 (d), 133.2 (s), 135.6 (s),
160.5 (s), and 168.0 (s); HRFAB MS (glycerol) [M + H]+, m/z
154.0509 (154.0505 calcd. for C7H8NO3). In the preparation of
[carbonyl-13C] AHBA hydrochloride, [carbonyl-13C] benzoic acid
was used as the starting compound. Other procedures were similar
to those for non-labeled AHBA hydrochloride preparation, then
we obtained [carbonyl-13C] AHBA hydrochloride (1.2 g, 38%)
by using [carbonyl-13C] benzoic acid (2.0 g). In the structure
elucidation of [carbonyl-13C] AHBA hydrochloride, the 1H-NMR
spectrum (CD3OD, 400 MHz) was consistent with that of non-
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