Sorangiadenosine, a new sesquiterpene adenoside from the myxobacterium Sorangium cellulosum

Article abstract of DOI:10.1021/ol800061h

Sorangiadenosine (1), a novel nucleoside possessing a bicyclic, eudesmane-type sesquiterpene as an auxiliary component, was isolated from the culture broth of the gliding bacterium Sorangium cellulosum KM1003, collected from Korean soil. The chemical structure of this compound was determined by combined spectroscopic and chemical analyses. The new compound exhibited moderate antibacterial activity against a wide range of bacterial strains.

Full text of DOI:10.1021/ol800061h

ORGANIC
LETTERS
2008
Vol. 10, No. 6
1167-1169
Sorangiadenosine, a New Sesquiterpene
Adenoside from the Myxobacterium
Sorangium cellulosum
Jong-Woong Ahn,† Kyoung Hwa Jang,‡ Soon-Chun Chung,§ Ki-Bong Oh,*,§ and
Jongheon Shin*,‡
DiVision of Marine EnVironment and Bioscience, College of Ocean Science
and Technology, Korea Maritime UniVersity, #1 Dongsam, Youngdo, Busan
606-791, Korea, Natural Products Research Institute, College of Pharmacy,
Seoul National UniVersity, #28 Yungun, Jongro, Seoul 110-460, Korea, and
School of Agricultural Biotechnology, Seoul National UniVersity, San 56-1,
Sillim, Gwanak, Seoul 151-921, Korea
shinj@snu.ac.kr
Received January 10, 2008
ABSTRACT
Sorangiadenosine (1), a novel nucleoside possessing a bicyclic, eudesmane-type sesquiterpene as an auxiliary component, was isolated from
the culture broth of the gliding bacterium Sorangium cellulosum KM1003, collected from Korean soil. The chemical structure of this compound
was determined by combined spectroscopic and chemical analyses. The new compound exhibited moderate antibacterial activity against a
wide range of bacterial strains.
The myxobacteria produce a wide variety of structurally
unique secondary metabolites.1 Several compounds derived
from these organisms exhibit diverse and potent biological
activity and are thus attracting a great deal of attention in
both biomedicine and synthetic chemistry.2,3 We have
previously reported the chemical structures and bioactivities
of bithiazole compounds isolated from Myxococcus fulVus.4,5
In the present study, we utilized a strain of Sorangium
cellulosum (number KM1003), the crude organic extract of
which exhibited significant antibacterial activity toward a
series of microbial strains. Here, we describe the chemical
structure and bioactivity of sorangiadenosine (1), a sesquit-
erpene adenoside of an unusual structural class, isolated from
this crude extract (Figure 1). Isolation and fermentation of
microbial strains were carried out according to the procedure
reported elsewhere.6
The bacterial strain KM1003 was isolated from a soil
sample collected from a dry field in Ansan, Korea using
WCX solid medium (CaCl2‚2H2O 0.1%, cycloheximide 50
µg/mL, agar 1.5%, pH 7.2). The strain was repeatedly
acclimatized using KAN-4 solid medium (CaCl2‚2H2O 0.1%,
cycloheximide 50 µg/mL, kanamycin sulfate 0.025%, agar
1.5%, pH 7.2) and maintained in VY/2 solid medium
(Baker’s yeast 0.5%, CaCl2‚2H2O 0.1%, cyanocobalamin 0.5
† Korea Maritime University.
‡ College of Pharmacy, Seoul National University.
§ School of Agricultural Biotechnology, Seoul National University.
(1) Reichenbach, H.; Gerth, K.; Kunze, B.; Hoefle, G. Trends Biotechnol.
1988, 6, 115-121.
(2) Reichenbach, H.; Hoefle, G. Biotechnol. AdV. 1999, 11, 219-277.
(3) Reichenbach, H.; Hoefle, G. In Drug DiscoVery from Nature; Grabley,
S., Thiericke, R., Eds.; Springer: New York, 1999; pp 149-179.
(4) Ahn, J.-W.; Woo, S.-H.; Lee, C.-O.; Cho, K.-Y.; Kim, B.-S. J. Nat.
Prod. 1999, 62, 495-496.
(5) Ahn, J.-W.; Jang, K. H.; Yang, H.-C.; Oh, K.-B.; Lee, H.-S.; Shin,
J. Chem. Pharm. Bull. 2007, 55, 477-479.
(6) Reichenbach, H.; Dworkin, M. In The Prokaryotes, 2nd ed.; Balows,
A., Truper, H. G., Dworkin, M., Harder, W., Schleifer, K.-H., Eds.;
Springer: New York, 1991; pp 3416-3487.
10.1021/ol800061h CCC: $40.75
© 2008 American Chemical Society
Published on Web 02/21/2008

Table 1. NMR Assignments for Compound 1a
no.
δH
δC
HMBC
1
2
1.40, m; 1.28, m
1.59, br d (13.8);
1.51, m
40.3 CH2
18.8 CH2 C-10
3
2.36, ddd (13.4, 13.1,
4.1);
2.05, br d (13.1)
36.7 CH2
57.8 C
4
5
2.48, dd (12.4, 2.0)
47.7 CH
C-4, C-6, C-7, C-10,
C-14, C-15
6
7
1.57, m; 1.33, m
1.89, br dd (11.9,
11.9)
26.0 CH2 C-10
45.5 CH
Figure 1. Chemical structures of sorangiadenosine (1).
8
9
1.54, m; 1.49, m
1.40, m; 1.31, m
26.7 CH2
44.8 CH2 C-5, C-7, C-10
34.2 C
µg/mL, agar 1.5%, pH 7.2), respectively. The strain was
identified as Sorangium cellulosum by morphological, bio-
chemical, and cultural characteristics.7,8 The slant culture of
KM1003 was cultivated in 2 L Erlenmeyer flasks containing
400 mL of a medium consisting of potato starch 0.8%,
glucose 0.2%, soyameal 0.1%, MgSO4‚7H2O 0.1%, CaCl2‚
2H2O 0.1%, and EDTA Fe(III)-Na+ salt 0.0008% in distilled
water. Also added to the medium was XAD-16 (1.5% w/v)
for the enhancement of metabolite production. Prior to
autoclaving, the pH of the medium was adjusted to 7.2 with
KOH. The flasks were incubated at 30 °C for 10 days on a
rotary shaker at 160 rpm.
10
11
12
13
14
15
2′
4′
5′
6′
8′
149.9 C
1.64, s
4.66, br s; 4.61, br s
0.96, s
1.39, s
8.21, s
20.6 CH3 C-7, C-11, C-13
108.5 CH2 C-7, C-11, C-12
19.2 CH3 C-1, C-5, C-9, C-10
20.5 CH3 C-3, C-4, C-5
151.5 CH
148.0 C
120.3 C
154.3 C
139.6 CH
88.0 CH
C-4′, C-6′
8.33, s
C-4′, C-5′, C-1′′
C-4′, C-8′, C-2′′,
C-3′′, C-4′′
C-1′′
C-1′′, C-5′′
C-5′′
1′′ 5.86, d (6.3)
At the end of fermentation (20 L), wet cell mass and
adsorbent resin XAD-16 were harvested by centrifugation
and extracted with acetone (1 L × 3). The aqueous layer,
after removal of acetone under vacuum, was adjusted to pH
7 by KOH and twice extracted with EtOAc (1 L × 2).
The organic solution was dried with Na2SO4, and the
solvent was evaporated under vacuum. The brown residue
was separated between 15% aqueous MeOH and n-hexane.
Guided by the results of antimicrobial tests and 1H NMR
analyses, the aqueous MeOH layer (670 mg) was directly
separated by C18 reversed-phase HPLC (YMC ODS-A
column, 20% aqueous MeOH) to afford pure compound 1
(28.4 mg).
2′′ 5.34, dd (6.3, 5.3)
3" 4.15, dd (5.3, 2.4)
4′′ 3.95, dt (2.4, 2.4)
5′′ 3.67, dd (12.6, 2.4);
3.54, dd (12.6, 2.4)
73.4 CH
70.6 CH
85.8 CH
61.6 CH2 C-3′′, C-4′′
NH 6.28, br s
C-4, C-5, C-10, C-15,
C-2′, C-5′, C-6′
a Data were obtained in DMSO-d6 solutions. Assignments were aided
by a combination of 1H COSY, TOCSY, gHSQC, and gHMBC experiments.
NMR spectra and corresponding isolated proton signals at
far downfield in the H NMR spectra, supported by the
Compound 1 was obtained as a pale-yellow gum, [R]20
-78.7 (c 1.17, MeOH). The absorption bands of its IR
1
D
o
presence of several nitrogens in FABMS data, suggested that
the heteroaromatic unit of 1 was indeed a base or related
moiety. The confirmation of this interpretation and the
identification of the base to be adenosine was accomplished
by a comparison of the NMR data with those of bases in the
literature as well as the gHMBC experiment in which several
key correlations were found between the protons at δ 8.33
and 8.21 with neighboring carbons.
spectrum suggested the presence of a hydroxyl and/or an
amino (3400 cm-1) function groups. The molecular formula
C25H38N5O4 was indicated by the positive HRFABMS at m/z
472.2922 [M + H]+ (calcd for C25H38N5O4, 472.2924).
The 13C NMR data of this compound showed signals of
25 carbons; 6 × C, 8 × CH, 8 × CH2, and 3 × CH3 (Table
1). A preliminary examination of the 1H, 13C, and 1H COSY
NMR data suggested that this compound was composed with
three distinct units: a heteroaromatic moiety, a pentose sugar,
and a hydrocarbon-type sesquiterpene. Observations of five
downfield carbons in the region of δ 155-120 in the 13C
The presence of a pentose sugar consisted of five
characteristic signals in the region of δ 88-60 in the 13C
NMR spectra, coupled with the corresponding six proton
signals in δ 5.9-3.0 in the 1H NMR spectra, was supported
by a combination of 1H COSY and TOCSY experiments in
which a linear spin system consisted of all of these protons
was traced. The sugar unit was identified as a D-ribose on
the basis of acid-hydrolysis followed by GC analysis and
(7) McCurdy, H. D. In Bergey’s Manual of DeterminatiVe Bacteriology;
Buchanan, R. E.; Gibbons, N. E., Eds.; Williams & Wilkins: Baltimore,
1974, pp 76-98.
(8) The strain is currently designated KCTC 11228BP and on deposit in
the Korean Collection for Type Cultures (KCTC) in Korea Research Institute
of Bioscience and Biotechnology (KRIBB), Taejon, Korea.
1168
Org. Lett., Vol. 10, No. 6, 2008

comparison of retention time with authentic pentoses.9,10
Compound 1 (1.5 mg) was dissolved in MeOH (1 mL) and
0.5 N KOH (0.5 mL) and stirred at room temperature for 2
h. After addition of saturated NaCl (3 mL), the aqueous layer
was extracted with EtOAc (3 mL × 3). The EtOAc-soluble
fraction contained a mixture of terpene compounds. The
aqueous layer was passed through an adsorption column of
HP-20 (0.5 × 10 cm) eluted with H2O (5 mL) and then with
MeOH (5 mL). The fraction eluted with MeOH afforded the
nucleoside moiety.
The nucleoside moiety (0.7 mg) was dissolved in 0.5 M
HCl/MeOH (0.5 mL) and heated at 65 °C for 15 h in a sealed
tube. After evaporation of the solvent by a stream of nitrogen,
the residue was dissolved in pyridine (50 µL) and treated
with hexamethyldisilazane (10 µL) and trimethylsilyl chloride
(5 µL) at room temperature for 30 min. Solvent was removed
by a nitrogen stream, and the residue dissolved in hexane
was analyzed by GC using an HP-1 column (3.2 mm × 50
m). Temperatures of injector and detector were 200 °C for
both, and a temperature gradient system was used for the
oven. The initial temperature was maintained at 30 °C for 3
min, ramped to 300 °C at the rate of 10 °C/min, then
maintained at 300 °C for 3 min. Peak of the hydrolysate
was detected at 19.17 min. Retention times for authentic
samples after being treated simultaneously with HMDS/
TMSCl were 19.17, 19.21 (D- and L-ribofuranose), 18.86,
18.89 (D- and L-lyxofuranose), 18.75, 18.78 (D- and L-
arabinofuranose), and 20.07, 20.10 min (D- and L-xylofura-
nose), respectively. Co-injection of the hydrolysate with the
authentic silyated D- and L-ribofuranose enhanced the peak
of D-ribofuranose only.
revealed several proton spin systems consisted of upfield
methylene and methine protons. Long-range correlations of
carbons bearing these protons with protons of the terminal
isopropylene and two methyls at δ 1.39 and 0.96 in gHMBC
experiment identified the sequiterpene moiety to be the
bicyclic 4-substituted eudesmane (Table 1). The stereochem-
istry at the asymmetric carbon centers at C-4, C-5, C-7, and
C-10 was assigned by NOESY experiments. Cross-peaks at
H-2â (δ 1.51)/H-14, H-2â/H-15, H-6â (δ 1.33)/H-8â (δ
1.49), H-6â/H-14, H-6â/H-15, H-8â/H-14, H-14/H-15 which
include several 1,3-diaxial correlations of the H-14 and H-15
methyl protons with neighboring protons showed the axial
orientations for these. Contrarily, another series of cross-
peaks at H-1R (δ 1.28) /H-3R (δ 2.36), H-1/H-5, H-1/H-9R
(δ 1.31), H-3/H-5, H-5/H-7, H-5/H-9R showed the axial
orientation of these protons at the opposite side of the decalin
plane. Thus the ring juncture and configurations were
assigned to be trans and 4 R*, 5 R*, 7 R*, 10R*, respectively.
Finally, the linkage between the adenine and ribose moiety
to form an adenosine was achieved by long-range correlations
of the H-8′ and H-1′′ with carbons in the alternative moiety.
Similarly, the linkage between the C-4 of eudesmane and
10-NH of the adenine was secured by long-range correlations
of the latter proton with several carbons at the sesquiterpene.
Thus, the structure of sorangiadenosine (1) was determined
to be a new adenosine containing a sesquiterpene of the
eudesmane skeleton. Although numerous novel compounds
have been isolated from myxobacteria, sorangiadenosine is
the first nucleoside containing polyprenyl moiety as an
auxiliary unit to the best of our knowledge.
In our measurement of bioactivity against gram-positive
and gram-negative bacteria, compound 1 exhibited moderate
antibacterial activity with MIC values of 25, 12.5, 6.25, 6.25,
12.5, and >100 µg/mL against the test strains Staphyloccus
aureus ATCC6538p, Bacillus subtilis ATCC 6633, Micro-
coccus leuteus IFC 12708, Proteus Vulgaris ATCC 3851,
Salmonella typhimurium ATCC 14028, and Escherichia coli
ATCC 25922, respectively. Further bioactivity and physi-
ological role of 1 are currently under investigation and will
be reported in due course.
The small vicinal coupling constants (J < 7 Hz) among
these protons revealed the furanose nature of this sugar
moiety that was secured by gHMBC experiments in which
a long-range coupling was obtained between the anomeric
proton at δ 5.86 and an oxymethine carbon at δ 85.8. In
addition, the â-orientation of the anomeric position was
assigned on the basis of characteristic 1JCH value (163.3 Hz)
as well as the strong enhancement (47%) between H-1 and
H-4 in selective-NOE experiment. Thus, the sugar moiety
was identified to be a â-D-ribofuranose.9,11,12
Acknowledgment. We express gratitude to Basic Science
Research Institute, Seoul, Korea, for providing mass data.
K.H.J. and S.-C.C. are the recipients of a fellowship from
the Ministry of Education, Korea, through the Brain Korea
21 Project. This work was partially supported by a grant from
the Korea Research Foundation (KRF-2006-005-J00502).
The sesquiterpene portion of 1 was also determined by a
combination of NMR experiments. Signals of protons and
carbons of a double bond (δC 149.9, 108.5; δH 4.66, 4.61)
1
and a vinyl methyl group (δC 20.6; δH 1.64) in the H and
13C NMR data readily revealed the presence of an isopro-
pylene moiety which corresponded to the head of a sesquit-
erpene. A combination of 1H COSY and TOCSY data
Supporting Information Available: Full experimental
procedures including spectroscopic and analytical data of
(9) Kobayashi, J.; Doi, Y.; Ishbashi, M. J. Org. Chem. 1994, 59, 255-
257.
1
compounds along with copies of the H NMR, 13C NMR,
(10) Shin, J.; Lee, H.-S.; Woo, L.; Rho, J.-R.; Seo, Y.; Cho, K. W.;
Sim, C. J. J. Nat. Prod. 2001, 64, 767-771.
(11) Chenon, M. T.; Pugmire, R. J.; Grant, D. M.; Panzica, R. P.;
Townsend, L. B. J. Am. Chem. Soc. 1975, 97, 4627-4636.
(12) Searle, P. A.; Molinski, T. F. J. Nat. Prod. 1994, 57, 1452-1454.
and 2D NMR spectra of compounds 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
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