Studies on terpenoids produced by actinomycetes: Oxaloterpins A, B, C, D, and E, diterpenes from Streptomyces sp. KO-3988

Article abstract of DOI:10.1021/np070326m

Following screening for terpenoids produced by Streptomyces sp. KO-3988, five new diterpenes named oxaloterpins A (1), B (2), C (3), D (4), and E (5) together with the known viguiepinone (6) were isolated from culture broth, and their structures were esta

Full text of DOI:10.1021/np070326m

1712
J. Nat. Prod. 2007, 70, 1712–1717
Studies on Terpenoids Produced by Actinomycetes: Oxaloterpins A, B, C, D, and E, Diterpenes
from Streptomyces sp. KO-3988
Keiichiro Motohashi,† Ryoko Ueno,† Masayuki Sue,† Kazuo Furihata,‡ Takuo Matsumoto,§ Tohru Dairi, Satoshi Omura,| and
Haruo Seto*,†
Faculty of Applied Bioscience, Tokyo UniVersity of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan, Graduate School of Agricultural and Life
Sciences, UniVersity of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan, AdVanced Technology Research Laboratories, Daiichi Sankyo Co.,
Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan, The Kitasato Institute, Minato-ku, Tokyo 108-8642, Japan, and Biotechnology Research
Center, Toyama Prefectural UniVersity, Toyama 939-0398, Japan
ReceiVed July 7, 2007
Following screening for terpenoids produced by Streptomyces sp. KO-3988, five new diterpenes named oxaloterpins A
(1), B (2), C (3), D (4), and E (5) together with the known viguiepinone (6) were isolated from culture broth, and their
structures were established on the basis of extensive NMR and MS analyses. The absolute configuration of oxaloterpin
A was determined by the modified Mosher’s method as 3R, 5S, 8S, 10R, 13S.
Terpenoids are the largest family of compounds found in nature,
with over 24 000 known examples.1,2 They are biosynthesized by
condensation of two types of C5 precursors, isopentenyl diphosphate
(IPP) and dimethylallyl diphosphate (DMAPP). IPP is a product
of the mevalonate pathway in eukaryotes and archaebacteria, while
it is synthesized through the recently discovered 2-C-methyl-D-
erythritol 4-phosphate (MEP) pathway in most bacteria, green algae,
and chloroplasts of higher plants.3 All Actinomycetes including
Streptomycetes utilize only the MEP pathway for the formation of
the primary metabolite IPP. On the other hand, some Actinomycetes
strains equipped with the MEP pathway machinery have been
shown to use the mevalonate pathway for production of secondary
terpenoidal metabolites as illustrated in Figure 1. These include
Kitasatosporia griseola (terpentecin producer),4 Actinoplanes sp.
A40644 (BE-40644 producer),5 Streptomyces sp. CL190 (naphterpin
A producer),6 Streptomyces sp. KO-3988 (furaquinocin A pro-
ducer),7 and Chainia rubra (napyradiomycin A producer).8 More
recently, Heide and co-workers reported that furanonaphthoquinone
I and endophenazine A, which are metabolites of Streptomyces
cinnamonensis, are produced via the mevalonate pathway.9 Because
these terpenoids showed interesting biological activities including
antitumor, antibacterial, and antioxidative properties, novel terpe-
noids produced by Actinomycetes were expected to be promising
candidates for drug discovery. Since such terpenoids have not been
well exploited, we carried out a screening of terpenoids from
Actinomycetes equipped with the mevalonate pathway gene cluster.
On the basis of our experimental results,10 we assumed that such
microorganisms possessed the mevalonate pathway gene cluster for
the production of secondary terpenoidal metabolites. In order to
efficiently select such microorganisms, we first investigated whether
the mevalonate pathway could be detected in the strains mentioned
above by Southern hybridization using the HMG-CoA reductase
gene as a probe. We found that Streptomyces sp. KO-3988, the
organism producing furaquinocin, possessed “two” corresponding
genes.10 Our later investigations showed that the organism had two
mevalonate pathway gene clusters, with one cluster being respon-
sible for production of antitumor agents, the furaquinocins.11
Subsequent gene analysis of the second mevalonate pathway gene
cluster showed that it was flanked by the GGDP (geranylgeranyl
diphosphate) synthase gene at its upstream region.12 Since GGDP
is the precursor of diterpenes, the strain was assumed to produce
diterpene compounds that had never been isolated from this strain.
We thus attempted to isolate diterpenes from the fermentation broth
of this strain and succeeded in purifying five new diterpenes as
well as the known viguiepinone (6).13
Results and Discussion
Streptomyces sp. KO-3988 was cultured at 28 °C for 7 days by
rotary shaking in 500 mL baffled Erlenmeyer flasks each containing
100 mL of the production medium used for furaquinocin.14 The
mycelial cake prepared from 30 L of fermentation broth was
extracted with acetone, and a small portion of the residue obtained
after removal of the solvent was analyzed by TLC developed with
n-hexane–EtOAc (1:1) or CHCl3–MeOH (20:1) and visualized with
vanillin–H2SO4. Spots that changed to bright purple or violet were
selected as candidates for terpenoids, and semipreparative purifica-
tion of the fractions corresponding to these spots was carried out
by Si gel column chromatography, preparative TLC, and C-18
RPHPLC. Samples thus prepared were subjected to 1H NMR
analysis, and fractions showing methyl proton singlets at around δ
* To whom correspondence should be addressed. Phone: +81-3-5477-
2321. Fax: +81-3-5477-2619. E-mail: haseto@nodai.ac.jp.
† Tokyo University of Agriculture.
‡ University of Tokyo.
§ Daiichi Sankyo Co.
1
1.0 in their H NMR spectra were assumed to contain terpenoids.
Toyama Prefectural University.
| The Kitasato Institute.
As a result of this screening, six fractions showing four methyl
10.1021/np070326m CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/31/2007

Studies on Terpenoids Produced by Actinomycetes
Journal of Natural Products, 2007, Vol. 70, No. 11 1713
Figure 1. Terpenoids biosynthesized via the mevalonate pathway.
1
proton singlets at around δ 1.0 in their H NMR spectra were
coupled to H-3, and another carbonyl at δC 167.6 (C-23) was
coupled to a methyl singlet at δH 2.25 (H-24) in the HMBC
spectrum. Furthermore, the broad singlet hydroxy proton (δH 10.08,
N-OH) was coupled to C-22 and C-23 in the HMBC spectrum.
These couplings could only be observed in the HMBC spectrum
taken at 4 °C when the hydroxy proton appeared as a sharp signal.
The connection between C-22 and C-23 through a nitrogen atom
was thus established, and finally C-21 and C-22 were connected
by elimination.
Direct evidence for these linkages was obtained from the EIMS
spectrum of 1; fragment peaks at m/z ) 343 and 358 were explained
by formation of the oxonium ions, as shown in Figure 3. This result
clearly established the presence of an oxalyl moiety and a unique
N,N-diacylhydroxylamine group in 1. The presence of this func-
tional group was corroborated by the IR spectrum (KBr) that
showed an absorption at 1800 cm-1 in addition to the expected
bands due to an ester carbonyl residue (1760 cm-1) and a hydroxy
group (3420 cm-1). The unusual band at 1800 cm-1 is compatible
with the value reported for N,N-diacylhydroxylamine (1799 cm-1).16
Thus the structure of 1 was established unequivocally. This is the
first report of the presence of the N,N-diacylhydroxylamine residue
in a natural product.
selected as plausible terpenoids. Two-dimensional NMR studies
on these components by COSY, HSQC, and HMBC as well as HR-
MS and IR measurements proved them to be terpenoids as expected,
and they were designated as oxaloterpins A (1), B (2), C (3), D
(4), and E (5). In addition, a known compound, viguiepinone (6),13
was isolated from this strain.
The HR-MS spectrum of oxaloterpin A (1) established its
molecular formula as C24H35NO5 (m/z 440.2407 [M + Na]+, calcd
440.2425), indicating 8 degrees of unsaturation. The 13C NMR and
HSQC spectra confirmed the presence of 24 carbons (Table 1),
including three carbonyl carbons [δC 167.6 (C-23), 158.0 (C-21),
and 152.7 (C-22)], four olefinic carbons [δC 150.3 (C-9), 150.1
(C-15), 116.5 (C-11), and 109.2 (C-16)], one oxymethine carbon
[δC 85.5 (C-3)], two methine carbons [δC 45.0 (C-5) and 29.4 (C-
8)], six methylene carbons [δC 41.5 (C-14), 38.3 (C-1), 37.6 (C-
12), 26.6 (C-7), 24.4 (C-2), and 18.2 (C-6)], five singlet methyl
groups [δC 27.7 (C-20), 25.3 (C-18), 22.5 (C-17), 18.2 (C-24), and
16.4 (C-19)], and three quaternary carbons [δC 38.4 (C-4), 37.4
(C-10), and 34.8 (C-13)]. Further structural information on 1
was obtained by analyzing HSQC, HMBC, and COSY spectra
(Figure 2).
The singlet methyl protons H-19 and H-20 showed 1H–13C long-
range couplings to C-3, C-4, and C-5 in the HMBC spectrum. The
singlet methyl protons H-17 were coupled to C-12, C-13, C-14,
and C-15, and the singlet methyl protons H-18 were coupled to
C-1, C-5, C-9, and C-10. The oxymethine proton H-3 was coupled
to C-1, C-2, C-4, C-19, and C-20. Long-range couplings were
observed between the methine proton H-5 and the following
carbons: C-4, C-6, C-7, C-10, C-18, C-19, and C-20. The olefinic
proton H-11 was coupled to C-8, C-9, C-12, and C-13. The
exomethylene proton H-16 was coupled to C-15. In addition, the
proton spin system from the methine proton H-8 to H-7 and H-14
was confirmed in the COSY spectrum. Furthermore, homoallylic
and allylic couplings were observed between H-8 and H-12 and
between H-8 and H-11, respectively. These results revealed the
presence of a viguiepinol skeleton15 in 1.
The molecular formula of oxaloterpin B (2) was established
as C24H38N2O4 by HR-MS measurement (m/z 419.2924 [M +
H]+, calcd 419.2904), indicating the presence in 2 of one more
nitrogen atom than in 1. Most of the NMR data of 2 were similar
to those of 1 (Table 1); the resonances for the N-acetyl group in
1 were, however, replaced by two methylene resonances (δC 62.5,
δH 3.75 for C-24 and δC 42.7, δH 3.46 for C-23), which were
ascribed to an HO–CH2CH2–N function. The 15N–1H HSQC
spectrum proved the presence of two secondary nitrogens (δN
99.5 and 69.1, coupled to protons at δH 7.11 and 8.08,
respectively) that were ascribed to a substituted urea function
(Figure 4) in order to explain 1H–13C long-range couplings
between H-23 and C-22 and between H-3 and C-21. This side-
chain structure was supported by HR-MS/MS data shown in
Figure 5. Among others, the strong product ion at m/z ) 87
(C3H7NO, m/z 87.0589 [M]+, calcd 87.0552) derived from the
precursor ion at m/z ) 149 (C4H9N2O4, 149.0597 [M + H]+,
calcd 149.0556) via the ion at m/z ) 131 (C4H7N2O3 131.0462
[M + H]+, calcd 131.0451) provided evidence for the substituted
urea function in 2. Other relevant ions were assigned to C3H6NO2
In addition to these resonances ascribed to the terpenoid moiety,
1 showed the following extra resonances, three carbonyls at δC
167.6 (C-23), 158.0 (C-21), and 152.7 (C-22), one methyl at δC
18.2 (C-24, δH 2.25, 3H, singlet), and one exchangeable OH proton
(δH 10.08). One of the carbonyls (δC 158.0, C-21) was long-range

1714 Journal of Natural Products, 2007, Vol. 70, No. 11
Motohashi et al.
Table 1. NMR Data (400 MHz, CDCl3) for Oxaloterpins A (1), B (2), C (3), D (4), and E (5) and Viguiepinone (6)
oxaloterpin A (1)
δH (J in Hz)
oxaloterpin B (2)
δH (J in Hz)
oxaloterpin C (3)
position
δC, mult.
δC, mult.
δC, mult.
δH (J in Hz)
1
2
3
4
38.3, CH2
24.4, CH2
85.5, CH
38.4, qC
1.95, 1.42, m
1.81, 1.73, m
4.67, dd, (11.6, 4.4)
38.3, CH2
24.6, CH2
83.9, CH
38.1, qC
1.91, 1.38, m
1.72, m
4.42, dd, (8.3, 7.8)
38.3, CH2
24.4, CH2
85.2, CH
38.3, qC
1.94, 1.41, m
1.80, 1.72, m
4.67, dd, (11.7, 3.9)
5
6
7
8
45.0, CH
18.2, CH2
26.6, CH2
29.4, CH
150.3, qC
37.4, qC
116.5, CH
37.6, CH2
34.8, qC
1.51, m
45.0, CH
18.1, CH2
26.6, CH2
29.3, CH
150.4, qC
37.5, qC
116.4, CH
37.4, CH2
34.8, qC
1.48, m
45.0, CH
18.1, CH2
26.5, CH2
29.3, CH
150.1, qC
37.5, qC
116.4, CH
37.5, CH2
34.8, qC
1.50, m
1.78, 1.58, m
1.68, 1.24, m
2.26, m
1.79, 1.58, m
1.67, 1.22, m
2.28, m
1.79, 1.58, m
1.67, 1.24, m
2.26, m
9
10
11
12
13
14
15
5.36, t, (2.4)
2.02, 1.77, m
5.34, t, (2.4)
2.00, 1.77, m
5.35, t, (2.2)
1.99, 1.77, m
41.5, CH2
150.1, CH
1.39, 1.02, m
5.79, dd,
(17.6, 10.7)
4.85, dd,
(10.4, 1.0)
4.91, dd,
(17.6, 1.0)
0.94, s
41.5, CH2
150.2, CH
1.39, 1.01, m
5.80, dd,
(17.2, 10.4)
4.87, dd,
(10.8, 1.0)
4.90, dd,
(16.8, 1.0)
0.94, s
41.4, CH2
150.1, CH
1.38, 1.02, m
5.79, dd,
(17.6, 10.7)
4.88, dd,
(10.7, 1.0)
4.92, dd,
(17.1, 1.0)
0.94, s
16
109.2, CH2
109.2, CH2
109.2, CH2
17
18
19
20
21
22
23
24
22.5, CH3
25.3, CH3
16.4, CH3
27.7, CH3
158.0, qC
152.7, qC
167.6, qC
18.2, CH3
22.4, CH3
25.2, CH3
16.2, CH3
27.7, CH3
153.9, qC
154.1, qC
42.7, CH2
62.5, CH2
22.4, CH3
25.3, CH3
16.4, CH3
27.7, CH3
158.2, qC
153.9, qC
1.09, s
1.02, s
0.87, s
1.07, s
0.93, s
0.88, s
1.09, s
1.07, s
0.87, s
3.46, q, (4.9)
3.75, dt, (5.8, 4.9)
8.08 br, t, (5.8)
7.11 br, s
2.25, s
NH
NH
NOH
10.08, s
9.49 br, s
oxaloterpin D (4)
H (J in Hz)
oxaloterpin E (5)
H (J in Hz)
viguirpinone (6)
δC, mult. H (J in Hz).
position
δC, mult.
δ
δC, mult.
δ
δ
1
2
3
4
38.3, CH2
24.4, CH2
84.9, CH
38.4, qC
1.93, 1.41, m
1.80, 1.71, m
4.66, dd, (11.9, 4.1)
38.5, CH2
24.8, CH2
81.9, CH
38.2, qC
1.88, 1.43, m
1.70, 1.67, m
4.35, dd, (11.6,; 4.4)
39.7, CH2
35.1, CH2
216.9, qC
47.9, qC
2.11, 1.64, m
2.72, 2.24, m
5
6
7
8
45.0, CH
18.1, CH2
26.6, CH2
29.4, CH
150.4, qC
37.5, qC
116.4, CH
37.6, CH2
34.8, qC
1.50, m
45.0, CH
18.1, CH2
26.7, CH2
29.3, CH
150.7, qC
37.5, qC
116.1, CH
37.5, CH2
34.8, qC
1.50, m
46.7, CH
18.6, CH2
26.7, CH2
29.3, CH
149.8, qC
37.5, qC
117.0, CH
37.5, CH2
34.9, qC
1.81, m
1.77, 1.42, m
1.66, 1.23, m
2.26, m
1.77, 1.57, m
1.67, 1.24, m
2.27, m
1.72, 1.67, m
1.73, 1.29, m
2.29, m
9
10
11
12
13
14
15
5.35, t, (2.4)
2.00, 1.75, m
5.33, t, (2.4)
2.00, 1.75, m
5.42, t, (2.4)
2.02, 1.77, m
41.5, CH2
150.2, CH
1.38, 1.01, m
5.83, dd,
(17.5, 10.7)
4.85, dd,
(10.7, 1.0)
4.93, dd,
(17.6, 1.0)
0.94, s
41.5, CH2
150.3, CH
1.39, 1.00, m
5.79, dd,
(17.6, 10.4)
4.88, dd,
(10.4, 1.2)
4.92, dd,
(17.6, 1.2)
0.93, s
41.8, CH2
150.0, CH
1.42, 1.05, m
5.80, dd,
(17.6, 10.8)
4.87, dd,
(10.8, 1.2)
4.94, dd,
(17.6, 1.2)
0.93, s
16
109.2, CH2
109.1, CH2
109.2, CH2
17
18
19
20
21
22
22.4, CH3
25.3, CH3
16.4, CH3
27.7, CH3
158.4, qC
159.8, qC
22.4, CH3
25.3, CH3
16.3, CH3
27.6, CH3
157.2, qC
22.4, CH3
24.1, CH3
24.9, CH3
21.8, CH3
1.09, s
1.02, s
0.88, s
1.06, s
0.89, s
0.89, s
1.26, s
1.09, s
1.05, s
23
24
NH
NH
NOH
6.88 br, s
5.79 br, s
4.65 br, s
4.65 br, s
(m/z 88.0854 [M]+, calcd 88.0398), CH4NO2 (m/z 62.0476 [M]+,
calcd 62.0242), and C3H4NO (m/z 70.0706 [M]+, calcd 70.0293).
Oxaloterpins C (3) and D (4) were also isolated from mycelium
extract as a colorless oil and white needles, respectively. The
molecular formulas of 3 and 4 were established as C22H33NO4 (m/z
398.2299 [M + Na]+, calcd 398.2307) and C22H33NO3 (m/z
382.2355 [M + Na]+, calcd 382.2358), respectively, by HR-MS
measurements. The 13C NMR data of 3 and 4 were similar to those
of 1 except for the absence of signals corresponding to the acetyl
group (C-23 and C-24) in 1 (Table 1). Two broad singlet NH

Studies on Terpenoids Produced by Actinomycetes
Journal of Natural Products, 2007, Vol. 70, No. 11 1715
ance of the side chain at C-3 with its downfield chemical shift being
δC 216.9. Thus 6 was proved to be a diterpene with a keto function
at C-3 and identified as viguiepinone (6).13
The absolute configuration of oxaloterpin A was established by
application of the modified Mosher’s method17 to a free alcohol
prepared by hydrolysis of 4. This alcohol was assumed as
viguiepinol (7) by detailed NMR studies and based on the X-ray
structure of 5. The relative configuration of 7 was established by
NOESY experiments as summarized in Figure 7. A methine proton
H-5 showed NOEs to H-3, H-8, and H-19, and from H-8 to H-17.
Singlet methyl proton H-18 showed a NOE to singlet methyl proton
H-20.
The absolute configuration of 7 was established by preparation
of its MTPA esters that were subjected to analysis by the modified
Mosher’s method. The differences of chemical shift values obtained
by subtracting (R)-MTPA ester from (S)-MTPA ester [δ∆ ) δ(S)-
MTPA – δ(R)-MTPA] are shown in Figure 8. On the basis of the
data obtained, the absolute configuration of 7 was concluded to be
3R, 5S, 8S, 10R, and 13S. This structure was in agreement with
that of viguiepinol (7) of plant origin determined by X-ray analysis.
The configurations of 1, 2, 3, 4, 5, and 6 were determined on
the basis of the absolute configuration of 7.
Figure 2. HMBC and COSY correlations observed for oxaloterpin
A (1). Bold lines show HMBC correlations observed with methyl
protons.
Antibacterial Activity. The biological activity of oxaloterpin
A (1) was investigated using more than 200 assay systems at MDS
Pharma services (Taipei, Taiwan, http://www.mdsinc.com ). Of the
tests conducted so far, 1 showed antibacterial activity against
Bacillus subtilis ATCC 43223 and Staphylococcus aureus ATCC
29213 with IC values of 1.9 and 3.7 nM/mL, respectively. Due to
a paucity of material, biological activities of other members of the
oxaloterpins were not tested.
Figure 3. Fragments of oxaloterpin A (1) observed by EIMS.
In conclusion, six diterpene compounds have been isolated from
a culture broth of the strain Streptomyces sp. KO-3988. We revealed
the structures of oxaloterpins A to E as having an oxalyl residue
or its analogue. Among them, the N-acetyl-N-hydroxyoxalylamide
residue present in oxaloterpin A is unique and has never before
been observed in a natural product. Furthermore we succeeded in
isolating viguiepinone (6), an oxidation product of viguiepinol; the
production of this alcohol had been suggested by heterologous
expression in E. coli of genes relevant to the biosynthesis of
pimaradiene from geranylgeranyl diphosphate of Streptomyces sp.
KO-3988.18
Figure 4. 1H–15N HSQC, 1H–13C HMBC, and 1H–1H COSY
correlations observed for oxaloterpin B (2). Values represent 13C
NMR chemical shifts, and those in brackets and parentheses show
1
15N and H NMR chemical shifts, respectively.
protons observed at δH 5.79 and 6.88 in the 1H NMR spectrum of
4 indicated a primary amide function. Thus, the structure of 4 was
determined to have an oxalyl amide function. As a consequence,
since 3 possessed one more oxygen atom than 4, it was concluded
to have an N-hydroxyoxalyl amide residue.
Experimental Section
The molecular formula of oxaloterpin E (5) was established as
C21H33NO2 from HR-MS data (m/z 663.5185 [2M + H]+, calcd
663.5095). The 13C NMR data of 5 were similar to those of 4 except
for the disappearance of a carbonyl resonance corresponding to C-22
in 4 with an upfield shift of C-3 in 5. These spectroscopy changes
together with the remaining unit (CONH2) were explained by the
presence of a carbamoyl ester side chain in 5. It may be necessary
to comment on the unusual MS data of 5. Although the molecular
ion peak (M + H or M + Na) was clearly observed with other
members of the oxaloterpins, only the dimeric ion peak was detected
with 5 in the complete absence of the corresponding monomeric
ion peak. Thus, although very unlikely, there remained the
possibility that this compound had a dimeric structure. X-ray
diffraction analysis was thus performed to confirm the structure of
5. The result shown in Figure 6 revealed that the compound had a
monomeric form and that the carbamoyl groups formed strong
intermolecular hydrogen bonds. The dimeric ion peak observed in
MS measurement is considered to be caused by this strong
intermolecular hydrogen bonding. The relative configuration of the
diterpene moiety was also determined by this analysis as 3R*, 5S*,
8S*, 10R*, 13S*.
General Experimental Procedures. Optical rotations were recorded
on a JASCO DIP-1000 polarimeter. IR spectra were obtained with a
JASCO 470 plus FTIR or a Shimadzu 8300 FTIR spectrometer. Both
1
1D H and 13C NMR spectra were recorded on a JEOL Alpha 400
1
NMR spectrometer operating at 400 MHz for H and 100 MHz for
13C. Two-dimensional 1H–1H COSY, NOESY, 1H–13C HSQC, HMBC,
1
and H–15N HSQC spectra were recorded on a JEOL Alpha 500 or a
Varian Inova 500 NMR spectrometer. Samples were dissolved in
CDCl3, and the solvent peak was used as an internal standard (δH 7.24
and δC 77.0). Ammonia (δN 0.00) was used as an external standard for
15N NMR spectra. High-resolution FAB, EI, and ESI mass spectra were
obtained using a JEOL HX-100 or a JEOL JMS-SX/SX-102A or an
Applied Biosystems MDS SCIEX Q-STAR LC-MS.
HPLC purifications were carried out using a Senshu PAK PEGASIL
ODS column (20ꢀ × 250 mm, at flow rate of 14.0 mL/min) equipped
with a Hitachi High Technologies L-2450 diode array detector. Merck
Si gel 60 F254 plastic-backed sheets were used for TLC analysis.
Preparative TLC was performed using Merck Si gel 60 F254 glass-backed
sheets. The detection for preparative TLC was carried out using color
reactions with vanillin–H2SO4.
Cultivation of Strain KO-3988. Streptomyces sp. KO-3988 was
cultivated in 15 mL test tubes each containing 5 mL of a preliminary
seed medium consisting of starch 1.0%, polypeptone 1.0%, molasses
1.0%, and meat extract 1.0% (pH 7.2). The test tubes were shaken on
a reciprocal shaker (200 rpm) at 28 °C for 2 days. Aliquots (1 mL) of
the broth were transferred to 500 mL baffled Erlenmeyer flasks
containing 100 mL of a production medium consisting of starch 2.0%,
Compound 6, which was obtained as a colorless oil, was shown
to have a molecular formula of C20H30O on the basis of HR-MS
measurement (m/z 287.2372 [M + H]+, calcd 287.2369). Detailed
analysis of HSQC and HMBC spectra of 6 revealed the disappear-

1716 Journal of Natural Products, 2007, Vol. 70, No. 11
Motohashi et al.
Figure 5. Fragment ions of oxaloterpin B (2) observed by LC-MS/MS.
acetone for 2 h, and the mycelium residue was removed by filtration.
The extract was evaporated in Vacuo to remove the acetone, and the
aqueous residue was extracted with EtOAc. The organic layer was dried
over anhydrous Na2SO4 and evaporated to dryness.
Purification of Oxaloterpins A (1), B (2), C (3), D (4), and E (5)
and Viguiepinone (6). The brown oil (30 L) was subjected to Si gel
column chromatography developed with n-hexane–EtOAc (1:1) to give
fractions containing 3, 4, and 6, and then with EtOAc–MeOH (3:1) to
give fractions containing 1, 2, and 5. The combined fraction containing
1, 2, and 5 was further purified by preparative TLC (CHCl3–MeOH,
20:1) to give separate bands corresponding to 1, 2, and 5. These
fractions were finally purified by HPLC. Detection of the compounds
was carried out at UV 200 nm. The column was eluted with MeCN in
H2O (90%) to give 1, 2, and 5 in this order. These fractions were
separately concentrated in Vacuo to give pure 1 (white powder, 24.0
mg), 2 (colorless oil, 2.5 mg), and 5 (colorless oil, 2.1 mg). The
combined fraction containing 3 and 4 was purified on a Si gel column
with CHCl3–MeOH (20:1) as the solvent system. Finally, pure samples
of 3 (colorless oil, 4.5 mg) and 4 (white needles, 10.8 mg) were obtained
with ODS HPLC eluted with MeCN in H2O (80%). The fraction
containing 6 was purified by Si gel column chromatography using
n-hexane–ether (5:1) as the solvent system. Fractions containing 6 as
a major component were combined and purified by ODS HPLC using
MeCN (100%) to yield a pure sample of 6 (colorless oil, 3.2 mg).
Conversion of 4 to Viguiepinol (7). Concentrated ammonia was
added dropwise to a MeOH solution of 4 (4.8 mg/ 0.5 mL), and the
solution was allowed to stand at room temperature for 2 h. The reaction
mixture was then evaporated to give a crude oil, which was purified
by preparative TLC with n-hexane–EtOAc (1:1) to afford free alcohol
7 as a colorless oil.
Figure 6. Intermolecular hydrogen bond of oxaloterpin E (5)
observed by X-ray diffraction analysis.
Preparation of MTPA Esters. To a solution of 7 containing a
catalytic amount of DMAP in CH2Cl2–pyridine (1:1) (1 mL) was added
(S)- or (R)-MTPA chloride (40 equiv), and the solution was then stirred
at room temperature for 24 h. The reaction mixture was then
concentrated to dryness, and the dry residue was extracted with 10 mL
of Et2O. The Et2O layer was then washed twice with 5 mL of acidic
H2O (pH 3) and then with 5 mL of H2O (pH 7) and finally washed
twice with 5 mL of 5% NaHCO3 solution. The organic layer was dried
over Na2SO4 and concentrated in Vacuo. The oily residue was purified
by ODS HPLC developed with 100% MeCN.
Figure 7. NOESYs observed for viguiepinol (7).
X-ray Crystal Data for 6. Crystal data for 6 were as follows:
colorless crystal, C21H33NO2, fw 331.50, orthorhombic, crystal size 0.20
× 0.10 × 0.10 mm, space group P212121, a ) 6.2395(7) Å, b )
13.6771(15) Å, c ) 22.794(3) Å, V ) 1945.2(4) Å3, Z ) 4, Dcalcd
)
1.132 g cm-3, F000 ) 728.00, 22 210 collected reflections, 3542 unique
reflections (Rint ) 0.027), final R1 ) 0.0372 (wR2 ) 0.0459) for 22 210
reflections with I > 2.00σ(I). The X-ray measurements were made on
a Rigaku RAXIS RAPID imaging plate area detector with graphite-
monochromated Cu KR (λ 1.54187 Å) radiation. The structure was
solved by direct methods (SIR92) and refined with full-matrix least-
squares on F and expanded using Fourier techniques. The non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were refined using
the riding model.
Figure 8. δ∆ values obtained for the MTPA esters of viguiepinol
(7).
soybean meal 1.0%, NaCl 0.3%, and CaCO3 0.3% (pH 7.0). The
fermentation was carried out at 28 °C and agitation at 170 rpm. After
7 days, the fermentation broth was separated into a mycelial cake and
filtrate by suction filtration. The mycelial cake was dipped in 60%

Studies on Terpenoids Produced by Actinomycetes
Journal of Natural Products, 2007, Vol. 70, No. 11 1717
Oxaloterpin A (1): white powder; [R]25D –14 (c 1.50, CHCl3); IR
(KBr) 3420, 1800, 1760, 1720 cm-1; 1H NMR (400 MHz, CDCl3) and
13C NMR (100 MHz, CDCl3), see Table 1; HR-MS m/z 440.2407 (calcd
for C24H35NO5Na, [M + Na]+ 440.2425).
and Ms. H. Suda of Chugai Pharmaceutical for recording HR-MS
spectra and helpful discussions on the interpretation of MS data.
1
Supporting Information Available: H NMR, 13C NMR, COSY,
Oxaloterpin B (2): colorless oil; [R]25 –19 (c 0.03, CHCl3); IR
HSQC, HMBC, HRMS, and IR spectra of oxaloterpins (1–5) and
viguiepinone (6) are available free of charge via the Internet at http://
pubs.acs.org.
D
(KBr) 3350, 1730, 1710 cm-1; H NMR (400 MHz, CDCl3) and 13C
1
NMR (100 MHz, CDCl3), see Table 1; HR-MS m/z 419.2924 (calcd
for C24H39N2O4, [M + H]+ 419.2904).
Oxaloterpin C (3): colorless oil; [R]25 –14 (c 0.23, CHCl3); IR
D
References and Notes
1
(KBr) 3400, 1740, 1700 cm-1; H NMR (400 MHz, CDCl3) and 13C
(1) Connolly, J. D.; Hill, R. A. Dictionary of Terpenoids; Chapman &
Hall: New York, 1992.
(2) Dewick, P. M. Nat. Prod. Rep. 2002, 19, 181–222.
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9500.
(6) Shin-ya, K.; Furihata, K.; Hayakawa, Y.; Seto, H. Tetrahedron Lett.
1990, 31, 6025–6026.
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1990, 55, 1132–1133.
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Umezawa, H. J. Antibiot. 1987, 40, 1740–1745.
(9) Bringmann, G.; Haagen, Y.; Gulder, T. A. M.; Gulder, T.; Heide, L.
J. Org. Chem. 2007, 72, 4198–4204.
(10) Kuzuyama, T.; Takahashi, S.; Dairi, T.; Seto, H. J. Antibiot. 2002,
55, 919–923.
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H.; Dairi, T. J. Bacteriol. 2006, 188, 1236–1244.
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Y.; Omura, S. J. Antibiot. 1990, 43, 247–252.
(15) Soriano-Garcia, M.; Guerrero, C.; Toscano, R. A. Acta Crystallogr.
1986, C42, 729–731.
(16) Ames, D. E.; Grey, T. F. J. Chem. Soc. 1955, 631–636.
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NMR (100 MHz, CDCl3), see Table 1; HR-MS m/z 398.2299 (calcd
for C22H33NO4Na, [M + Na]+ 398.2307).
Oxaloterpin D (4): white needles (MeOH); mp 205 °C; IR (KBr)
1
3450, 1740, 1690 cm-1; H NMR (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3), see Table 1; HR-MS m/z 382.2355 (calcd for
C22H33NO3Na, [M + Na]+ 382.2358).
Oxaloterpin E (5): colorless oil; [R]25 –15 (c 0.18, CHCl3); IR
D
1
(KBr) 3430, 1730, 1700 cm-1; H NMR (400 MHz, CDCl3) and 13C
NMR (100 MHz, CDCl3), see Table 1; HR-MS m/z 663.5185 (calcd
for C42H67N2O4, [2M + H]+ 663.5095).
1
Viguiepinone (6): colorless oil; IR (KBr) 1730, 1710 cm-1; H
NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3), see Table
1; HR-MS m/z 287.2372 (calcd for C20H31O, [M + H]+ 287.2369).
Viguiepinol (7): colorless oil; 1H NMR (400 MHz, CDCl3) δ 5.80
(1H, dd, J ) 17.2, 10.4 Hz, H-15), 5.34 (1H, t, J ) 2.4 Hz, H-11),
4.93 (1H, dd, J ) 17.6, 1.0 Hz, H-16), 4.87 (1H, dd, J ) 10.7, 1.0 Hz,
H-16), 3.21 (1H, dd, J ) 10.5, 5.0 Hz, H-3), 2.26 (1H, m, H-8), 2.00
(1H, m, H-12), 1.87 (1H, m, H-1), 1.78 (1H, m, H-6), 1.75 (1H, m,
H-12), 1.66 (1H, m, H-7), 1.62 (1H, m, H-2), 1.58 (1H, m, H-6), 1.38
(1H, m, H-5), 1.36 (1H, m, H-14), 1.28 (1H, m, H-1), 1.22 (1H, m,
H-7), 1.05 (3H, s, H-18), 1.00 (1H, m, H-14), 0.96 (3H, s, H-19), 0.94
(3H, s, H-17), 0.85 (3H, s, H-20); 13C NMR (100 MHz, CDCl3) δ 151.0
(C, C-9), 150.3 (CH, C-15), 116.0 (CH, C-11), 109.1 (CH2, C-16), 79.1
(CH, C-3), 44.8 (CH, C-5), 41.6 (CH2, C-14), 38.9 (CH2, C-1), 38.9
(C, C-4), 37.6 (C, C-10), 37.6 (CH2, C-12), 34.9 (C, C-13), 29.4 (CH,
C-8), 28.1 (CH2, C-2), 27.8 (CH3, C-20), 26.8 (CH2, C-7), 25.3 (CH3,
C-18), 22.5 (CH3, C-17), 18.3 (CH2, C-6), 15.3 (CH3, C-19).
(18) Ikeda, C.; Hayashi, Y.; Itoh, N.; Seto, H.; Dairi, T. J. Biochem. (Tokyo)
2006, 141, 37–45.
Acknowledgment. This work was supported by a Grant-in-Aid for
Scientific Research (B) from the Ministry of Education, Science, Sports
and Culture to H.S. (14360067). The authors thank Dr. N. Nakayama
NP070326M