Ieodoglucomides A and B from a marine-derived bacterium Bacillus licheniformis

Article abstract of DOI:10.1021/ol300202z

Ieodoglucomides A (1) and B (2), unique glycolipopeptides consisting of an amino acid, a new fatty acid, a succinic acid, and a sugar, were isolated from a Marine-derived bacterium Bacillus licheniformis. The absolute stereochemistry of 1 and 2 was determined by deploying coupling constants, Marfey's and Mosher's methods, and literature reviews. Compounds 1 and 2 displayed moderate in vitro antimicrobial activity. Furthermore, ieodoglucomide B (2) exhibited cytotoxic activity against lung cancer and stomach cancer cell lines with GI₅₀ values of 25.18 and 17.78 μg/mL, respectively.

Full text of DOI:10.1021/ol300202z

ORGANIC
LETTERS
2012
Vol. 14, No. 6
1464–1467
Ieodoglucomides A and B from a Marine-
Derived Bacterium Bacillus licheniformis
Fakir Shahidullah Tareq,†,‡ Ji Hye Kim,‡ Min Ah Lee,‡ Hyi-Seung Lee,‡ Yeon-Ju Lee,‡
Jong Seok Lee,‡ and Hee Jae Shin*,†,‡
Department of Marine Biotechnology, University of Science and Technology,
217 Gajungro, Yuseong-gu, Daejeon, Republic of Korea, and Marine Natural Products
Chemistry Laboratory, Korea Ocean Research & Development Institute, Ansan,
Republic of Korea
shinhj@kordi.re.kr
Received January 26, 2012
ABSTRACT
Ieodoglucomides A (1) and B (2), unique glycolipopeptides consisting of an amino acid, a new fatty acid, a succinic acid, and a sugar, were isolated
from a Marine-derived bacterium Bacillus licheniformis. The absolute stereochemistry of 1 and 2 was determined by deploying coupling
constants, Marfey’s and Mosher’s methods, and literature reviews. Compounds 1 and 2 displayed moderate in vitro antimicrobial activity.
Furthermore, ieodoglucomide B (2) exhibited cytotoxic activity against lung cancer and stomach cancer cell lines with GI50 values of 25.18 and
17.78 μg/mL, respectively.
Glycolipids have been identified as membrane receptors
of various pathogenic microorganisms, principally bac-
teria and viruses as well as antineoplastic agents during the
past few years.1,2 Several types of two-chain and geminal
glycolipids have been described in the literature.3,4 We
report herein two novel glycolipopeptides, ieodogluco-
mides A (1) and B (2), possessing a new skeleton from a
marine sediment bacterium Bacillus licheniformis.
B. licheniformis is a saprophytic Gram-positive bacteri-
um which has been devoted typically in the fermentation
industry for the rendering of amylases, proteases, antibio-
tics, and special chemicals with low risk of adverse effects
to humans and the environment.5 Several groups also
accounted for biosurfactants, antibiotics, and antifungal
(polypeptide) compounds from this strain originated from
terrestrials.6,7 Marine bacteria have been proven to be
potential producers of bioactive compounds with unique
structural characteristics, since the biodiversity of the
marine environment is higher than that of the terrestrial
environment.8,9 Thus, the strain 09IDYM23, identified
as B. licheniformis by 16s rRNA sequencing (GenBank
accession no. JQ349055) and showing antimicrobial activ-
ity, was further advanced to chemical examination.
The producing strain 09IDYM23 was isolated from
a sediment sample collected from Ieodo, Republic of
Korea’s southern reef where salt concentration and pH
† University of Science and Technology, Republic of Korea.
‡ Korea Ocean Research & Development Institute.
(5) He, L.; Chen, W.; Liu, Y. Microbiol. Res. 2006, 161, 321–326.
(6) Sung-Chyr, L.; Mark, A. M.; Sharma, M. M.; Georgioul, G.
Appl. Environ. Microbiol. 1994, 60, 31–38.
(1) Karlsson, K. A. Annu. Rev. Biochem. 1989, 58, 309.
(2) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2754and references cited therein.
(7) Thaniyavarn, J.; Rossngsawang, N.; Kameyama, T.; Haruki, M.;
Imanaka, T.; Morikawa, M.; Kanaya, S. Biosci. Biotechnol. Biochem.
2003, 67, 1239–1244.
(3) (a) Lockhoff, O. Angew. Chem., Int. Ed. Engl. 1991, 30, 1611 and
references cited therein. (b) Zhang, Z.; Fukunaga, K.; Shinizu, T.;
Nakao, K. Carbohydr. Res. 1995, 277, C1–C3 and references cited
therein.
(8) Fenical, W. Chem. Rev. 1993, 93, 1673–1683.
(4) Castro, M. J. L.; Kovensky, K.; Fernandez Cirelli, A. Tetrahedron
Lett. 1997, 38, 3995.
(9) Fenical, W.; Jensen, P. R. In Marine Biotechnology; Attaway, D.,
Zaborsky, O., Ed.; Plenum Press: New York, 1993; Vol. 1, pp 419ꢀ457.
r
10.1021/ol300202z
Published on Web 02/24/2012
2012 American Chemical Society

of water were 32.3 (g/L) and 8.01, respectively. Thus, the
growth conditions of the strain were adjusted by varying
pH, temperature, and salt concentration of seawater be-
fore proceeding to large-scale cultures. It was found from
the findings that the productivity and growth of the strain
was significant at salt concentration 18.3 g/L, pH 7.02,
and temperature 32 °C. In fact, the physiological process
of bacteria altered at diverse conditions.10 Cultures of the
unicellular bacterium were then grown following the above
conditions and fermentation broth (50 L) was extracted with
ethyl acetate. The chromatographic separation of the ethyl
acetate extract using ODS open column in combination with
semipreparative MPLC and HPLC resulted in the isolation
of two novel glycolipopeptides, ieodoglucomides A and B.
Table 1. 1H and 13C NMR and HMBC Data of 1 in CD3OD
no.
δH, mult (J in Hz)
δC
HMBC
HMA
1
176.3
173.2a
36.9
2
2.22, t (7.5)
1.61, m
1, 3
3
27.0
4ꢀ11
12
13
14
15
16
17
Ala
10
1.29, brs.
1.29, brs.
1.46, m
29.9ꢀ30.9
26.6
32.2
3.42, m
85.6
12, 13, 1000
14
1.87, m
32.3
0.93, d (6.5)
0.93, d (6.5)
18.3
14, 15
14, 15
18.7
176.3
174.3a
49.3
47.3a
17.8
20
4.37, q (7.0, 14.5)
1, 10
30
1.38, d (7.5)
8.05, d (9.0)a
10, 20
1, 20, 30
NH
SA
100
Ieodoglucomide A (1) was isolated as a yellowish oil. Its
molecular formula was assigned as C30H53NO12 with the
174.0
171.8a
30.2
200
300
400
2.61, m
2.61, m
100
100, 400
1
aid of H NMR, 13C NMR data and HRESI-MS at
[M ꢀ H]ꢀ m/z 618.3497 (calcd 619.3568 for C30H53NO12).
The IR spectrum showed absorption bands for hydroxyl
(3349 cmꢀ1) and amide carbonyl (1660 cmꢀ1) groups. The
1H NMR spectrum of 1 (Table 1) in CD3OD showed
signals for one methyl proton at δ 1.38 (H-30) and one
methine proton at δ 4.37 (H-20) and in DMSO-d6 an
additional signal for the amide proton at δ 8.05 (1H, d,
J = 9.0 Hz) was shown. Associated 13C NMR signals at δ
176.3 (C-10), 49.3 (C-20) and 17.8 (C-30) corresponding to
carboxylated proton, methine proton, and methyl proton
demonstrated the presence of Alanine (Ala) in the com-
pound 1. The 1H NMR spectrum also displayed signals for
two methylprotonsatδ 0.93(H-16and H-17), one methine
proton at δ 1.87 (H-15), an oxygenated methine proton at
δ 3.42 (H-14), and a signal appearing as a singlet at δ
1.29 for long alkyl chain, revealing the presence of a 14-
hydroxy-15-methylhexadecanoic acid (HMA) unit in the
molecule. The COSY, HMBC, and ROSEY correlations
(Figure 1) also established the structure of the HMA unit.
Carbon resonances also supported the existence of the
HMA unit in the compound 1, which was confirmed
by ESI-MS analysis [(M þ H)þ m/z at 286.19)] after acid
hydrolysis (Figure 2) of 1. Hexadecanoic acid with 14-
hydroxyl and 15-methyl groups has not been reported yet
in natural products. Thus, the established HMA is a new
29.9
175.9
173.2a
Glu
1000
2000
3000
4000
5000
6000
4.29, d (8.0)
3.18, dd (8.5, 12.7)
3.33, m
104.2
75.5
78.1
71.9
75.1
65.2
14
3000
4000
3.28, m
3000, 5000
3.40, m
4.18, dd (6.0, 12.5)
4.44, d (11.5)
4000, 100
a Chemical shifts and coupling constant were determined in
DMSO-d6.
75.5, and 104.2 revealing the presence of β-glucopyranose
(Glu) moiety11 as the sugar residue in 1.
Succinic acid (SA) moiety attached to the molecule was
determined by the presence of two carbonyl carbon signals
at δ 171.8 (C-100) and δ 173.2 (C-400) and two methylene
carbon signals at δ 29.9 (C-300) and δ 30.2 (C-200). HSQC
1
fatty acid from natural sources. In the H and 13C NMR
spectra of 1, ananomeric proton appearedat δ 4.29(H-1000)
and carbon signals resonated at δ 65.2, 71.9, 75.1, 78.1,
(10) Thomas, D. N.; Dieckmann, G. S. Science 2002, 295, 641–644.
(11) (a) Bock, K.; Pederson, C. In Advances in Carbohydrate Chem-
istry and Biochemistry; Jipson, R. S., Horfon, D., Eds.; Academic Press:
New York, 1983; Vol. 41, pp 27ꢀ46. (b) Mansoor, T. A.; Shinde, P. B.;
Luo, X.; Hong, J.; Lee, C. O.; Sim, C. J.; Son, B. W.; Jung, J. H. J. Nat.
Prod. 2007, 70, 1481–1486. (c) Pettit, G. R.; Tang, Y.; Knight, J. C.
J. Nat. Prod. 2005, 68, 974–978.
Figure 1. Key HMBC, COSY, and ROESY correlations of 1 in
CD3OD and DMSO-d6.
Org. Lett., Vol. 14, No. 6, 2012
1465

spectra revealed the attachments of these two methylene
carbons with protons resonated at δ 2.61 (m, overlapped).
Furthermore, HMBC correlations (Figure 1) between
H-200 and C-100 and between H-300 and C-400 confirmed
the existence of SA moiety in the molecule. Moreover,
these four distinct units were connected with the help
unit showed a correlation with a carbon signal at δ 174.1
(C-100) of SA unit. Thus, the structure of ieodoglucomide
B (2) was determined and the overall assignments of
1H and 13C NMR data were unambiguously made on
1
1
the basis of the Hꢀ H COSY, ROESY, HSQC, and
HMBC spectra.
Figure 3. ΔδH values (ΔδH = δS ꢀ δR) obtained for (S)- and
(R)-MTPA esters of HMA of 1.
The absolute stereochemistry of amino acid (AA), C-14
of HMA, and Glu moiety in 1 and 2 was determined by
Marfey’s method,12 Mosher’s method,13,14 and compar-
isons of optical rotation and Rf value with an authentic
sample, respectively. After acid hydrolysis, the reaction
mixtures were extracted with n-Hexane, ethyl acetate and
methanol to obtain HMA, AA, and Glu moiety respec-
tively. Ala of 1 was found to be of the L form. The absolute
configuration of the stereocenter at C-14 of HMA unit in 1
and 2 was determined by treating HMA with (R)-(ꢀ)- and
(S)-(þ)-R-methoxy-R-(trifluoromethyl)phenylacetyl chlo-
ride (MTPA-Cl) in dry pyridine separately to yield (S)- and
(R)- MTPA ester derivatives 1a, 1b, 2a, and 2b, respectively
(Figure 3 and Supporting Information). All proton signals
Figure 2. Acid hydrolysis of 1.
of HMBC and ROSEY correlations. The connectivity
between HMA and NH of Ala group was confirmed by
the ROESY and HMBC correlations between NH proton
and H-2 and C-1 of HMA unit, respectively. The oxyge-
nated proton at δ 3.42 (H-14) of HMA unit showed an
HMBC correlation with the anomeric carbon of Glu at δ
104.2 (C-1000). On the other hand, methylene proton signals
at δ 4.18 (H-6a000) and 4.44 (H-6b000) of Glu unit correlated
with a carbon signal at δ 174.0 (C-100) of SA unit. From
these detailed NMR data analyses, the planar structure of
ieodoglucomide A (1) was unambiguously determined.
Ieodoglucomide B (2) was also isolated as a yellowish oil
similar to 1, and its molecular formula was assigned to be
C29H51NO12 with the aid of HRESI-MS [M ꢀ H]ꢀ at
m/z 604.3339 (calcd 605.3411 for C29H51NO12) and 1H and
13C NMR analysis (Table 1). The IR spectrum showed
absorption bands for hydroxyl (3349 cmꢀ1) and amide
carbonyl (1660 cmꢀ1) groups. The NMR results were
found to be essentially identical to those of 1 which con-
firmed that 2 also contained HMA, SA and Glu moiety.
The only difference was the observation of glycine (Gly)
instead of Ala which was also confirmed by the absence of
1H and 13C signals of methyl group and the existence of an
amide proton at δ 8.02 and a methylene proton as singlet at
δ 3.89 (H-20). The ROESY and HMBC data in DMSO-d6
indicated the correlations between NH proton of Gly with
H-2 and C-1 of HMA unit and with methine proton of Gly
at δ 3.89 (H-20). The oxygenated proton at δ 3.43 (H-14) of
HMA unit showed an HMBC correlation with the anome-
ric carbon of Glu at δ 104.2 (C-1000). Furthermore, methy-
lene protons at δ 4.17 (H-6a000) and 4.44 (H-6b000) of Glu
1
of the derivatives were assigned by 1Hꢀ H COSY experi-
1
ment and H NMR chemical shifts (ΔδH = δS ꢀ δR).
These data allowed the assignment of the absolute config-
uration of C-14 of 1 and 2 as S (Figure 3). The absolute
configuration of the Glu moiety for both compounds was
determined to be the β-D-form by comparing the signof the
optical rotation [0.6 mg, [R]23D þ27 (c 0.14, H2O)] and Rf
value (0.58) with an authentic sample.
Ieodoglucomides A and B were found to have moderate
antimicrobial activity when tested against both Gram-
positive and Gram-negative bacteria and fungi following
broth dilution assay technique (Table 2).15 Different
growth conditions of bacteria and fungi were maintained
while culturing these microorganisms.16,17
The cytotoxicity of 1 and 2 was evaluated against six
human cancer cell lines (breast cancer: MDA-MB-231,
colon cancer: HCT15, prostate cancer: PC-3, lung cancer:
NCI-H23, stomach cancer: NUGC-3, and renal cancer:
(12) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(13) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092–4096.
(14) Tsuda, M.; Toriyabe, Y.; Endo, T.; Kobayashi, J. Chem. Pharm.
Bull. 2003, 51, 448–451.
(15) Appendio, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.;
Stavri, M.; Smith, E.; Rahman, M. M. J. Nat. Prod. 2008, 71, 1427–1430.
(16) Oluwatuyi, M.; Kaatz, G. W.; Gibbons, S. Phytochemistry 2004,
65, 3249–3254.
(17) Yu, J. Q.; Lei, J. C.; Yu, H. D.; Cai, X.; Zou, G. L. Phytochem-
istry 2004, 65, 881–884.
1466
Org. Lett., Vol. 14, No. 6, 2012

Table 2. Minimum Inhibitory Concentrations (MICs) of 1 and 2
MICs (μg/mL)
Table 3. Human Cancer Cell Line Inhibition Values (GI50
expressed in μg/mL) of 1 and 2
cancer cell lines
1
2
ADRa
microorganisms
1
2
PCa
breast cancer: MDA-MB-231
colon cancer: HCT-15
prostate cancer: PC-3
lung cancer: NCI-H23
stomach cancer: NUGC-3
renal cancer: ACHN
>30
>30
>30
>30
>30
>30
>30
>30
>30
0.38
0.26
0.93
0.93
0.28
0.22
Gram-positive bacteria
S. aureus
8
8
2
2
2
B. subtilis
16
16
16
8
25.18
17.78
>30
B. cereus
Gram-negative bacteria
S. Typhi
16
8
16
16
8
2
2
2
E. coli
a ADR: adriamycin as standard.
P. aeruginosa
fungi
8
immunostimulative, and immunesuppessive activities.20
Some of these activities of glycolipids would appear pro-
mising for the treatment of Alzheimer’s disease.21 Accord-
ingly, we intend to advance further biological investigation
of 1 and 2 as these novel glycolipopeptides may have a
useful role against some diseases.
C. albicans
A. niger
32
32
32
16
4
4
a PC: positive control (azithromycin for bacteria and amphotericin B
for fungi).
ACHN) according to a sulforhodamine B (SBR) assay
(Table 3).18 Compound 2 exhibited cancer cell growth
inhibition against lung cancer (NCI-H23) and stomach
cancer (NUGC-3) cell lines, with GI50 values of 25.18 and
17.78 μg/mL, respectively.
In conclusion, to the best of our knowledge, ieodoglu-
comides A and B represent the first examples of novel
bioactive metabolites containing a new fatty acid unit
(HMA) from a marine-derived bacterium.
The moststructurallydistinctpartofieodoglucomidesA
and B is the presence of HMA, AA, and SA that possesses
a C30 and C29 skeleton with a glycolipid backbone which
has never been described before from natural sources.
Ieodoglucomides A and B bear a slight resemblance to
the well-known powerful immunostimulant R-galactosyl
ceramide (R-GalCer) KRN7000,19 but the overall
structures are completely unprecedented. It is also
noteworthy that some glycolipids are known to possess
antiviral, antifungal, antimicrobial, COX-2 inhibitory,
Acknowledgment. We are grateful to Dr. C. Kun,
Korea Basic Science Institute, Ochang, Republic of Korea,
for providing mass data and to Dr. Jieun Yun, Bioevalua-
tion Center, KRIBB, Republic of Korea, for carrying out
cytotoxicity tests. We also express gratitude to Professor
Dr. Choudhury Mahmood Hasan, Department of Phar-
maceutical Chemistry, University of Dhaka, Bangladesh,
for helping in part with chemical studies. This research was
supported in part by the Ministry of Land, Transport and
Maritime Affairs, Korea.
(18) Skehan, P.; Storeng, R; Scudiero, D.; Monks, A.; McMohan, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyed, M. R.
J. Natl. Cancer Inst. 1990, 82, 1107–1112.
(19) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.;
Sawa, E.; Yamaji, K.; Kobayashi, E.; Fukushima, H.; Koezuka, Y.
J. Med. Chem. 1995, 38, 2176–2187.
Supporting Information Available.
General proce-
dures, bioassay protocols, chemical derivatization, data
tables, and NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
(20) Pettit, G. R.; Tang, Y.; Knight, J. C. J. Nat. Prod. 2005, 68,
974–978.
The authors declare no competing financial interest.
(21) Tan, R. X.; Chen, J. H. Nat. Prod. Rep. 2003, 20, 509–534.
Org. Lett., Vol. 14, No. 6, 2012
1467