Sarmentosamide, a novel hexadienamide from Thai soil actinomycetes

Article abstract of DOI:10.1080/14786419.2012.671313

A new hexadienamide derivative named sarmentosamide (1) was identified from the culture of Streptomyces sp. SBI108 isolated from Thai soil under an herb. The structure was elucidated on the basis of spectroscopic data, and the absolute configuration was determined by chemical degradation.

Full text of DOI:10.1080/14786419.2012.671313

Natural Product Research, 2013
Natural Product Research
Vol. 27, No. 3, 226–231, http://dx.doi.org/10.1080/14786419.2012.671313
2
012, 1–6, iFirst
Sarmentosamide, a novel hexadienamide from Thai soil actinomycetes
a
Shigeru Kitani , Ayako Tomio , Akkaraphol Srichaisupakit , Ratama Daduang ,
a
b
a
b
bc
d
Bungonsiri Intra , Watanalai Panbangred , Naoya Oku , Yasuhiro Igarashi
d
ac
and Takuya Nihira *
a
International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka
5
Rd., Bangkok 10400, Thailand; MU-OU Collaborative Research Center for Bioscience and
b
65-0871, Japan; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI
c
Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand;
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu,
Toyama 939-0398, Japan
d
(
Received 21 January 2011; final version received 22 January 2012)
A new hexadienamide derivative named sarmentosamide (1) was identified from
the culture of Streptomyces sp. SBI108 isolated from Thai soil under an herb. The
structure was elucidated on the basis of spectroscopic data, and the absolute
configuration was determined by chemical degradation.
Keywords: hexadienamide; Streptomyces sp. SBI108; Piper sarmentosum Roxb
1
. Introduction
Natural products have a major commercial impact in the fields of medicine and
agriculture, where they continually give rise to novel applications and new modes of action
and targets. Actinomycetes produce an array of natural products in the form of secondary
metabolites, and are currently the most useful microorganisms for this purpose (Berdy,
2
005). We recently initiated a screening program to discover new natural products from
actinomycetes isolated from different ecosystems in Thailand (Igarashi et al., 2010).
During our extensive search for structurally unique secondary metabolites by HPLC/UV-
based chemical screening, Streptomyces sp. SBI108 was found to produce two antibiotics,
spectinabilin (Ishibashi, Nishiyama, Shizuri, & Yamamura, 1992) and tryptophan-
dehydrobutyrine diketopiperazine (Santamaria, Cabezas, & Avendano, 1999). To date,
a few hexadienamide derivatives such as erythrococcamide A and erythrococcamide C
from Dinosperma erythrococca (F. Muell.) (Latif, Hartley, Rice, Waigh, & Waterman,
1
998) and the numbing principle from Chrysanthemum morifolium Ramar (Shahat, Apers,
Pieters, & Vlietinck, 2001) have been reported. Erythrococcamide A represents a novel
class of isobutylamide, and shows insecticidal activity. The geometries of the double bonds
in the dien moiety of the erythrococcamide A and the numbing principle are assigned as
E,E and E,E, respectively. In this article, we report the isolation and structural elucidation
0
0
0
of a structurally novel hexadienamide compound, N-(4 -carbamoyl-3 -penten-2 -yl)hexa-
Z,4E-dienamide, which we have named sarmentosamide (1).
2
*
Corresponding author. Email: nihira@icb.osaka-u.ac.jp
ISSN 1478–6419 print/ISSN 1478–6427 online
*
Corresponding author. Email: nihira@icb.osaka-u.ac.jp
�
http://dx.doi.org/10.1080/14786419.2012.671313
2012 Taylor & Francis
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2
S. Kitani et al.
Natural Product Research
227
2
. Results and discussion
The producing strain, Streptomyces sp. SBI108, was newly isolated from a soil sample
collected under an herb plant (Piper sarmentosum Roxb.) in Bangkok, Thailand. This
microorganism was identified as Streptomyces sp. on the basis of its 16 S rRNA sequence.
Comparison of the 16S rRNA partial sequence (1391 bp) with sequences in the NCBI
database revealed that the closest phylogenetic neighbours of the strain were Streptomyces
T
spectabilis NBRC 15441 (AB184677, with a sequence identity of 99.8%) and
T
Streptomyces lusitanus strain NBRC 13464 (AB184424, sequence identity of 98.5%).
Some strains of Streptomyces spectabilis produce spectinabilin which is also detected in the
culture broth of Streptomyces sp. SBI108, and a Streptomyces lusitanus strain produces
tetracycline and chlortetracycline aureomycin. The culture supernatant of Streptomyces
sp. SBI108 was extracted with ethyl acetate, and subjected to solid-phase extraction with
reversed-phase resin. The 20% CH CN fraction was then purified by HPLC using two
3
different reversed-phase columns to yield 1 in pure form.
Compound 1 was obtained as a colourless amorphous solid. The high resolution EIMS
showed a molecular ion peak at m/z 222.1366, corresponding to a molecular formula of
1
3
C H N O , which implied the presence of five unsaturation sites in the structure. The
C
NMR spectrum confirmed 12 signals, indicating four aliphatic carbons, six carbons in the
1
2
18
2
2
1
olefinic region and two carbons in the carbonyl regions. The H NMR spectrum in
combination with the HSQC and DEPT-135 spectrum revealed proton signals for five
2
olefinic methines, three methyl groups, an aliphatic methine and three quaternary sp
1
carbons. The H– H COSY correlations demonstrated two fragments: H-2–H-3–H-4–H-
1
0 0 0
5
–H-6, suggesting the presence of a pentadiene moiety, and H-1 –H-2 –H-3 . Furthermore,
in the HMBC spectrum, two olefinic methine protons (H-2 and H-3) were coupled to the
carbonyl carbon C-1, which indicated a 2,4-hexadienoyl moiety. The geometries of the
C-2–C-3 and C-4–C-5 double bonds were assigned as Z and E, respectively, on the basis of
the vicinal coupling constants (11.8 and 15.1 Hz, respectively) and the NOEs between H-2
and H-3, H-3 and H-5, H-4 and H-6. On the other hand, in the HMBC spectrum of 1, the
0
0
aliphatic methine proton H-2 ’, the olefinic methine proton H-3 , and the methyl proton
0
0
H-6 were coupled with the quaternary carbon C-4 . Additionally, two proton signals from
0
0
0
H-3 and H-6 were also coupled to the carbonyl carbon C-5 . These correlation data
0
0
0
suggested the presence of a 2 -substituted-4 -methyl-3 -pentenyl moiety. An NOE between
0
0
0
0
H-6 and H-2 allowed an E configuration of the double bond between C-3 and C-4 .
Moreover, the IR spectrum was in coherence with the presence of amide and olefinic
�
1
functions (3330, 3241, 3175, 1645, 1632 and 1603 cm ), and the aliphatic methine proton
0
H-2 showed an HMBC correlation to the carbonyl carbon C-1 in the 2,4-hexadienoyl
0
0
0
moiety. Thus, the planar structure of 1 was elucidated as N-(4 -carbamoyl-3 E-penten-2 -
yl)hexa-2Z,4E-dienamide (Figure 1).
Figure 1. Sarmentosamide (1) (left) and the COSY and HMBC correlations (right).

2
28
S. Kitani et al.
Natural Product Research
3
The absolute configuration of the single chiral centre in 1 was determined by chemical
degradation of this molecule and application of Marfey’s analysis (Harada et al., 1996) to
the resulting alanine (Ala). Compound 1 was treated with RuCl –NaIO to cleave the
3
4
double bonds, and the diacid product was acid-hydrolysed to liberate Ala. After
derivatisation of the acid-hydrolysate with L-FDLA (N-(3-fluoro-4,6-dinitrophenyl)-L-
leucinamide), reversed-phase HPLC analysis was performed, revealing the presence of
0
L-Ala in the degradation product. Thus, it was concluded that the chirality of the 1 -methyl
0
group on the 2 -carbon shows an S-configuration (Figure 1).
Sarmentosamide (1) is an interesting amide that contains two rare structural motifs.
The unsaturated acyl chain present in 1 has been reported from a sponge (Tanaka & Higa,
1
996) and fungi (Fujimoto, Tsunoda, Uzawa, & Tatsuno, 1982; Kinsman et al., 1998) but
not from bacteria. The nitrogen-containing right-half portion of the molecule has never
been reported in a natural product, although a similar building block has been used in
synthetic chemistry (Murray, Sun, Turchi, Brown, & Gauthier, 1999).
As sarmentosamide (1) has an interesting skeleton, we attempted to examine the
�1
biological activity at the 50 mg disk level. However, compound 1 did not show any
activities against two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus),
a Gram-negative bacterium (Escherichia coli), a typical eukaryote (Saccharomyces
cerevisiae) and a plant pathogenic oomycete (Phytophthora sojae), while apramycin and
hygromycin as positive controls clearly showed biological activities against all the tested
microorganisms.
Sarmentosamide, a structurally unusual hexadienamide compound, is produced under
the same cultivation conditions with two known and structurally unrelated bioactive
compounds (spectinabilin and tryptophan-dehydrobutyrine diketopiperazine), which were
found in the initial purification step of sarmentosamide, and identified. Extensive efforts
on isolation and identification of secondary metabolites from actinomycetes can be
expected to lead to the discovery of additional chemically diverse natural products.
3
3
. Experimental
.1. General experimental procedures
The UV spectrum was recorded on a Hitachi U-3200 spectrophotometer. NMR spectra
1
were recorded on a JEOL JNM-ECP400 at 400 MHz. The H and C chemical shifts were
13
referenced to the solvent signal (�H 3.3 and �C 49.1 in CD OD). HREIMS data was
3
recorded on a JEOL JMS-700. Optical rotation was measured on a JASCO P-1020
polarimeter. The IR spectrum was recorded on a Shimadzu FTIR-8400 S.
3
.2. Bacterial material
The producer Streptomyces, designated as SBI108, was isolated from a soil sample under
an herb (P. sarmentosum) in Bangkok, Thailand. Suspensions from the soil sample diluted
with 0.9% NaCl solution were spread on water proline agar [proline 1.0% and agar 1.2%]
�1
�1
supplemented with nalidixic acid (25 mg mL ) and cycloheximide (50 mg mL ). After the
plates were incubated at 28 C for 14 days, colonies were transferred to Waksman agar
ꢀ
[
glucose 1.0%, peptone 0.5%, meat extract 0.5%, NaCl 0.3% and agar 1.2%] and
ꢀ
ꢀ
cultivated at 28 C for 10 days. The strain was kept at �80 C as a spore stock. A Genbank
search for the 16 S rRNA sequence (1341 bp, GenBank: HQ336791) from SBI108 showed
that this strain belonged to the genus Streptomyces. The Streptomyces strain was deposited
at the culture collection of the National Center for Genetic Engineering and
Biotechnology, Thailand.

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S. Kitani et al.
Natural Product Research
229
3
.3. Fermentation
Mycelium of Streptomyces sp. SBI108, which was grown on Seino’s agar slant [soluble
starch 1.0%, NZ amine type A 0.3%, meat extract 0.1%, yeast extract 0.1%, CaCO 0.3%
3
and agar 1.5% (pH 7.0)], was inoculated by using an inoculating needle in a 500-mL
baffled flask containing 100 mL of seed 301 medium consisting of soluble starch 2.4%,
glucose 0.1%, peptone 0.3%, meat extract 0.3%, yeast extract 0.5% and CaCO 0.4% (pH
3
�
7
.0). The flask was cultivated on a rotary shaker (160 rpm) for 2 days at 28 C. The seed
culture (2 mL) was transferred into 100 mL of the 301 medium in 500-mL baffled flasks,
�
and a total of 150 flaks were cultured on a rotary shaker (160 rpm) for 7 days at 28 C.
3
.4. Extraction and isolation
The fermentation broth (13.2 L) was separated into mycelia and supernatant by filtration
with gauze. The supernatant was extracted with an equal volume of ethyl acetate, and the
organic layer was evaporated as the crude extract. The crude extract (998 mg) was
3
subjected to reversed-phase column chromatography (Sep-Pak Vac 35 cm C cartridge;
1
8
Waters, Milford, MA) with a CH CN–H O step gradient system (0 : 1, 2 : 8 and 1 : 0 v/v).
3
2
The 20% CH CN fraction (351 mg) was subjected to reversed-phase HPLC using a
3
CAPCELL PAK C18 column (UG80; 4.6 i.d. ꢀ 250 mm; Shiseido, Tokyo, Japan)
developed with a linear gradient system [eluents: H O (A), CH CN (B); 0–5 min 15% B,
2
3
ꢁ1
5
2
–20 min 15% B to 40% B, 20–25 min 40% B to 85% B; flow 1 mL min ; UV detection at
54 nm], to give a partially purified compound 1 (t ¼ 17.5 min). Final purification was
R
conducted by repeated reversed-phase HPLC using an XTerra RP18 column (4.6
i.d. ꢀ 250 mm; Waters) developed with a linear gradient system [eluents: H O (A), CH CN
2
3
containing 0.1% formic acid (B); gradient 0 min 15%, 0–20 min 15% B to 40% B; flow
ꢁ
1
mL min ; UV detection at 254 nm], to yield 15.5 mg of sarmentosamide 1 eluted at
1
1
4.5 min in pure form.
3
.5. Sarmentosamide (1)
2
D
8
Colourless amorphous solid; ½�ꢂ ꢁ141 (c 0.05, MeOH); UV (MeOH) ꢀ
(log ") 206
max
ꢁ1
(
4.35), 259 (4.54); IR (KBr) ꢁmax 3330, 3241, 3175, 1645, 1632, 1603 cm ; HREIMS (þ)
1
3
m/z 222.1366 (Calcd for C H N O , 222.1368); C-NMR (100 MHz, CD OD): ꢂ 13.2
12 18 2 2 3
0 0 0
C-6 , CH ), ꢂ 18.6 (C-6, CH ), ꢂ 20.5 (C-1 , CH ), ꢂ 44.3 (C-2 , CH), ꢂ 119.1 (C-2, CH),
3 3 3
(
0
0
ꢂ 129.8 (C-4, CH), ꢂ 132.3 (C-4 , C), ꢂ 138.9 (C-5, CH), ꢂ 139.0 (C-3 , CH), ꢂ 142.6 (C-3,
0
1
CH), ꢂ 168.2 (C-1, C), ꢂ 174.2 (C-5 , C); H-NMR (400 MHz, CD OD): 1.24 (3 H, d,
3
0
0
0
J ¼ 7.0 Hz, H-1 ; HMBC with C-2 , 3 ), 1.82 (3 H, dd, J ¼ 6.8, 1.7 Hz, H-6; HMBC with C-
0
0
0
0
0
0
2
, 3, 4, 5), 1.92 (3 H, d, J ¼ 1.5 Hz, H-6 ; HMBC with C-1 , 2 , 3 , 4 , 5 ), 4.77 (1 H, dq,
0
0
0
0
J ¼ 8.8, 7.0 Hz, H-2 ; HMBC with C-1, 1 , 3 , 4 ), 5.55 (1 H, dd, J ¼ 11.8, 0.7 Hz, H-2;
HMBC with C-1, 3, 4, 6), 5.99 (1 H, qd, J ¼ 15.1, 6.8 Hz, H-5; HMBC with C-3, 6), 6.22
0
0
0
0
0
0
(
1 H, dq, J ¼ 8.8, 1.5 Hz, H-3 ; HMBC with C-1 , 2 , 4 , 5 , 6 ), 6.39 (1 H, dd, J ¼ 11.8,
1
C-3, 6).
1.4 Hz, H-3; HMBC with C-1, 4, 5), 7.39 (1 H, dd, J ¼ 15.1, 11.4 Hz, H-4; HMBC with
3
.6. RuCl -catalised oxidation and acid-hydrolysis of sarmentosamide (1)
3
Compound 1 (0.2 mg) and NaIO (2.7 mg) were completely dissolved with CH CN (40 mL)
4
3
and H O (30 mL). The solution was mixed with CCl (40 mL) and a buffered solution of
2
4
ꢁ
1
RuCl (40 mL, 1 mg mL in 0.1 M sodium phosphate, pH 7.6), and the reaction mixture
3
was vigorously stirred for 2 h. After removing the solvents in vacuo, the residue was
dissolved in 2 M HCl, and extracted with ethyl acetate. The organic layer was evaporated
in a vacuum hydrolysis tube, and 6 M HCl was added. The tube was deaerated, sealed and

2
30
S. Kitani et al.
Natural Product Research
5
�
heated at 110 C for 13 h. The HCl solution was removed under reduced pressure
�
at 70 C to give an acid hydrolysate, which was used for further analysis as described
below.
3
.7. Chirality determination of Ala derived from 1 by Marfey’s analysis
The acid hydrolysate in the previous section was added to a 0.1% (w/v) acetone solution of
L-FDLA [N-(3-fluoro-4,6-dinitrophenyl)-L-leucinamide] (50 mL) and 0.1 M NaHCO3
�
(
100 mL). After the reaction mixture was kept at 70 C for 1 h, it was neutralised with
0
.2 M HCl. An aliquot of the solution was analysed by reversed-phase HPLC using a
Cosmosil 5C -AR-II column (4.6 i.d. ꢀ 250 mm; Nacalai Tesque Inc., Kyoto, Japan)
1
8
developed with 18% aqueous CH CN containing 0.1% formic acid with detection at
3
ꢁ
1
40 nm and a flow rate of 1 mL min . The chromatographic standards for L-Ala and the
3
D-Ala equivalent were prepared by reacting L-Ala or D-Ala with L-FDLA, respectively,
with the same procedure. The retention times of FDLA-Ala (min) were: L-Ala (16.3),
D-Ala (22.0). The sample derived from compound 1 contained L-Ala (16.2).
3
.8. Biological assays
Compound 1 was tested in agar diffusion assays against Bacillus subtilis PCI219,
Staphylococcus aureus 209 P, E. coli ATCC25922, S. cerevisiae ATCC6275 and P. sojae
P6497 by two independent experiments. The pure compound showed no activity in these
assays.
4
. Conclusion
We successfully isolated and identified a new hexadienamide compound named
sarmentosamide from Thai soil actinomycetes, Streptomyces sp. SBI108. Sarmentosamide
(
1), which showed no biological activity under our conditions tested, has an interesting
skeleton that contains two rare structural motifs from bacteria: the 2Z,4E-unsaturated
acyl chain and the nitrogen-containing right-half portion, confirming the presence of
chemically diverse natural products from Thai soil actinomycetes.
Acknowledgements
This study was supported in part by the ‘Research Project in the Field of Biotechnology’ from the
Japan Society for the Promotion of Science, the National Research Council of Thailand and the
National Science and Technology Development Agency of Thailand to T.N., W.P. and Y.I., by a
‘FrontierLab@OsakaU’ scholarship from Osaka University to A.S, and by an RGJ scholarship
PHD/0314/2551 from the Thailand Research Fund to B.I.
References
Berdy, J. (2005). Bioactive microbial metabolites. The Journal of Antibiotics, 58, 1–26.
Fujimoto, Y., Tsunoda, H., Uzawa, J.,
& Tatsuno, T. (1982). Structure of lslandic acid, a new
metabolite from Penicillium islandicum Sopp. Journal of the Chemical Society, Chemical
Communications, 83–84.
Harada, K., Fujii, K., Hayashi, K., Suzuki, M., Ikai, Y., & Oka, H. (1996). Application of DL-FDLA
Derivatization to determination of absolute configuration of constituent amino acids in peptide by
advanced Marfey’s method. Tetrahedron Letters, 37, 3001–3004.
Igarashi, Y., Shimasaki, R., Miyanaga, S., Oku, N., Onaka, H., Sakurai, H., . . . Panbangred, W. (2010).
Rakicidin D, an inhibitor of tumor cell invasion from marine-derived Streptomyces sp. The Journal of
Antibiotics, 63, 563–565.

6
S. Kitani et al.
Natural Product Research
231
Ishibashi, Y., Nishiyama, S., Shizuri, Y., & Yamamura, S. (1992). Total synthesis of (þ)-isoaureothin:
Determination of the absolute configurations of aureothin, isoaureothin and spectinabilin. Tetrahedron
Letters, 33, 521–524.
Kinsman, O.S., Chalk, P.A., Jackson, H.C., Middleton, R.F., Shuttleworth, A., Rudd, B.A., . . . Hayes, M.V.
(1998). Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis
inhibition. The Journal of Antibiotics, 51, 41–49.
Latif, Z., Hartley, T.G., Rice, M.J., Waigh, R.D., & Waterman, P.G. (1998). Novel and insecticidal
isobutylamides from Dinosperma erythrococca. Journal of Natural Products, 61, 614–619.
Murray, W.V., Sun, S., Turchi, I.J., Brown, F.K., & Gauthier, A.D. (1999). Diels�Alder reactions of amino acid-
derived trienes. The Journal of Organic Chemistry, 64, 5930–5940.
Santamaria, A., Cabezas, N., & Avendano, C. (1999). Synthesis of tryptophan-dehydrobutyrine diketopiper-
azines and analogues. Tetrahedron, 55, 1173–1186.
Shahat, A.A., Apers, S., Pieters, L., & Vlietinck, A.J. (2001). Isolation and complete NMR assignment of the
numbing principle from Chrysanthemum morifolium. Fitoterapia, 72, 89–91.
Tanaka, J., & Higa, T. (1996). Zampanolide, a new cytotoxic marcrolide from a marine sponge. Tetrahedron
Letters, 37, 5535–5538.