Absolute configuration of iminimycin B, a new indolizidine alkaloid, from Streptomyces griseus OS-3601

Article abstract of DOI:10.1016/j.tetlet.2016.06.040

Iminimycin B, a novel indolizine alkaloid featuring a rare pyridinium, was isolated from the cultured broth of a streptomycin-producing strain, Streptomyces griseus OS-3601, through a physicochemical screening method. Its structure was elucidated on the basis of mass and NMR analyses. Stereochemical assignment of iminimycin B was archived by NMR studies, electronic circular dichroism (ECD) analysis, and advanced Marfey's method.

Full text of DOI:10.1016/j.tetlet.2016.06.040

Accepted Manuscript
Absolute configuration of iminimycin B, a new indolizidine alkaloid, from
Streptomyces griseus OS-3601
Takuji Nakashima, Rei Miyano, Hirotaka Matsuo, Masato Iwatsuki, Tatsuya
Shirahata, Yoshinori Kobayashi, Kazuro Shiomi, George A. Petersson, Yōko
Takahashi, Satoshi Ōmura
PII:
S0040-4039(16)30709-2
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.06.040
TETL 47767
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 April 2016
3 June 2016
10 June 2016
Please cite this article as: Nakashima, T., Miyano, R., Matsuo, H., Iwatsuki, M., Shirahata, T., Kobayashi, Y., Shiomi,
K., Petersson, G.A., Takahashi, Y., Ōmura, S., Absolute configuration of iminimycin B, a new indolizidine alkaloid,
from Streptomyces griseus OS-3601, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/j.tetlet.2016.06.040
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Absolute configuration of iminimycin B, a new indolizidine alkaloid, from
Streptomyces griseus OS-3601
Takuji Nakashima, Rei Miyano, Hirotaka Matsuo, Masato Iwatsuki, Tatsuya Shirahata, Yoshinori Kobayashi,
Kazuro Shiomi, George A. Petersson, Yōko Takahashi, Satoshi Ōmura
O
NH
HO
S
O
N+
Streptomyces griseus OS-3601 ꢀ

1
Tetrahedron Letters
jo u r n a l h o m e p a g e : w w w . e ls e v ie r . c o m
Absolute configuration of iminimycin B, a new indolizidine alkaloid, from
Streptomyces griseus OS-3601
Takuji Nakashima a, Rei Miyano b, Hirotaka Matsuo a, Masato Iwatsuki a,b, Tatsuya Shirahata c, Yoshinori
Kobayashi c, Kazuro Shiomi a,b, George A. Petersson d, Yōko Takahashi a and Satoshi Ōmura a
a Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-5791, Japan
b Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-5791, Japan
c School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-5791, Japan
d Hall-Atwater Laboratories of Chemistry, Wesleyan University, Middletown, CT 06459-0180, USA.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Iminimycin B, a novel indolizine alkaloid featuring a rare pyridinium, was isolated from the
cultured broth of a streptomycin-producing strain, Streptomyces griseus OS-3601, through a
physicochemical screening method. Its structure was elucidated on the basis of mass and NMR
analyses. Stereochemical assignment of iminimycin B was archived by NMR studies, electronic
circular dichroism (ECD) analysis, and advanced Marfey’s method.
Available online
2016 Elsevier Ltd. All rights reserved.
Keywords:
Pyridinium
Indolizidine
Iminimycin
Streptomyces
Natural products produced by microorganisms have been a
rich source for drug discovery. Many drugs developed from
natural products, such as leucomycin1 and avermectin2, have been
discovered by bioassay-guided isolation. Our screening program,
physicochemical (PC) screening, was allows to detect more
different types of natural products in a culture broth than
bioassay program.
maximum at 279 and 394 nm and molecular ion peak at m/z 399
was observed in the other ODS fraction without iminimycin A.
This compound, designated iminimycin B (1), was a new
indolizine alkaloid possessing
a N-acetyl-cysteine and a
pyridinium (Figure 1) instead of iminium moiety of iminimycin
A.
On our ongoing PC screening program, we has successfully
discovered mangromicins3 and new nanaomycin analogs4 from
actinomycete culture broths. Moreover, this screening program
has recently led to discover a new indolizidine alkaloid,
iminimycin A, from the culture broth of Streptomyces griseus
OS-3601. The strain OS-3601 was isolated as a streptomycin-
producing strain at Aso, Kumamoto-Prefecture, Japan in 1972,
and preserved for over 40 years.5
N+
Iminimycin A
6'
5'
O
10
NH
HO
1'
2'
1
2
S
9
14
16
12
8
3'
Many indolizine alkaloids with their unique structures and
biological activities were mainly isolated from metabolites
plants, frogs and ants.6 The alkaloid compounds have been also
found to produced by some actinomycete strains, but a few only
compounds are extant. Cyclizidine and its analog were isolated
from secondary metabolites of Streptomyces sp. and
Saccharopolyspora sp.,7 and indolizomycin was isolated from the
strain SK2-52 that was formed by protoplast fusion treatment
between non-antibiotic-producing mutants of S. griseus and S.
tenjimariensis.8 Iminimycin A is an indolizidine alkaloid,
consisting of an octahydroindolizine skeleton with a triene side
chain and a cyclopropane ring, and the most distinctive feature of
this compound is to have an unusual iminium group.5 In PC
screening for discovery of new compounds from the culture broth
of this strain, an unidentified compound with an absorption
O
15
N+
3
17
13
11
7
5
6
1
Figure 1. Structures of iminimycin A and 1.
Isolation of 1 was guided by physico-chemical properties such
as its molecular formula (C22H27N2O3S+) and UV spectrum (λmax
279 and 394 nm), using LC/UV and LC/MS equipment. A strain
of S. griseus OS-3601 was cultured in producing medium (100
mL × 100), for 5 days at 27 °C. The 5-day old culture broth (10
L) was centrifuged to separate cells and supernatant. The

2
Tetrahedron
supernatant subjected to Diaion HP-20 column and sequential
1
1
As shown in Figure 2, the H-1H COSY and H-13C HMBC
spectra indicated the presence of five partial structures: C-1/C-
2/C-10 to form a cyclopropane ring, C-5/C-6, C-3/C-11−C-14, C-
16/C-17, C-2’/C-3’. 1H-13C HMBC Analysis confirmed the
presence of a pyrrolidinium ring fused with a cyclopropane,
based on the correlations from H2-10 to C-1, C-2, C-3, and C-9,
and from H-2 to C-1, C-3 and C-9. An indolizidinium ring was
identified, based on HMBC correlations from H-5 to C-3, C-7
and C-9, from H-6 to C-8, from H-7 to C-5, C-6 and C-9, and
from H-3 to C-9 (Figures S7 and S8). A characteristic band at
1388, 1481 and 1605 cm-1 in the IR spectra of 1 suggests a
pyridine moiety (Figure S1). Moreover, the HMBC correlations
from H-11 to C-13, from H-13 to C-11, C-12 and C-15, from H-
14 to C-12, C-15 and C-16, from H-16 to C-17 and C-15-Me,
from H3-15-Me to C-14, C-15 and C-16 confirmed the presence
of a 5-methyl-hepta-1,3,5-trienyl unit. All geometries of this
triene were determined as E based on large coupling constants
(Table 1) and ROESY correlations (H-13/H3-15-Me and H-14/H-
16) (Figure S10). The HMBC correlations from H-3 to C-11and
H-12, from H-11 to C-2 and C-3, from H-12 to C-3 revealed that
this triene unit was attached to the C-3 position. Remaining
signals were classified into one heteroatom-bound methylene at
δH 3.39 and 3.78 (dd, J = 4.5, 14.0), δC 37.0; one heteroatom-
bound methine at δH 4.44 (dd, J = 4.5, 7.0), δC 55.0; two carbonyl
carbons at δC 175.5 and δC 173.0; and one methyl group at δH
ODS flash column chromatography. Eventually, 1 was purified
by reversed-phase HPLC (Scheme S1).
24.6
Iminimycin B (1) was isolated as a white powder ([α]D
-
13.9 (c = 0.1, MeOH). The HR-ESIMS of 1 produced the M+ ion
at m/z 399.1736 indicating the molecular formula was
C22H27N2O3S+ (calculated value for C22H27N2O3S+, 399.1737).
The 1H and 13C NMR spectral data of 1 are shown in Table 1.
Table 1. 1H and 13C NMR Data of Iminimycin B (1) in
CD3OD
Positi
13C ppm,
mult
1H ppm, mult, Hz
on
1
2
3
5
6
7
8
9
10a
10b
11
12
13
14
15
15-
24.9, CH
23.2, CH
78.0, CH
138.6, CH
126.1, CH
145.3, CH
137.5, qC
159.5, qC
17.7, CH2
3.23, m
2.46, m
5.59, d (9.5)
8.42, d (6.0)
7.73, dd (6.0, 8.0)
8.53, d (8.0)
1.03, m
1.77, m
5.88, dd (9.5, 15.5)
6.73, m
6.21, dd (11.0, 15.5)
6.51, d (15.5)
127.4, CH
139.8, CH
124.6, CH
143.7, CH
135.8, qC
1
1.97 (3H, s), δC 22.8. From these detailed analysis of H-1H
1
COSY and H-13C HMBC spectra, the presence of N-acetyl-
cysteine moiety was determined (Figure 1). Since a long-range
1H-13C heteronuclear correlation was observed from H2-3’ (δH
3.39, 3.78) to an aromatic quaternary carbon C-8 (δC 137.5), we
confirmed that N-acetyl-cysteine moiety was connected at C-8
through a sulfur atom, which is also implied by the molecular
formula. The relative configuration of 1 was determined as
1S*,2R*,3S* by ROESY spectrum that gave cross peaks for H-
3/H-5, H-3/H-10, and H-2/H-12 supported by no coupling
11.9, CH3
1.77, s
Me
16
17
1'
2'
3' a
131.4, CH
14.2, CH3
175.5, qC
55.0, CH
37.0, CH2
5.74, q (6.5)
1.77, d (6.5)
4.44, dd (4.5, 7.0)
3.39, dd (7.0, 14.0)
3.78, dd (4.5, 14.0)
3' b
between H-2 and H-3 (Figure 3). The NMR, UV
and IR
5'
6'
173.0, qC
22.8, CH3
spectra of 1 were similar to louludinium chloride, whish is an
indolizine alkaloid with pyridinium ion, from a marine bluegreen
alga, Lyngbya gracilis.9
1.97 (3H, s)
1
The H NMR spectrum of 1 in CD3OD displayed eight sp2
methine protons from 8.53 to 5.74 ppm, along with two allylic
methyl groups (both δH 1.77), four sp3 methines (δH 5.59, 4.44,
3.23, and 2.46), two methylenes (δH 1.03 and 1.77, 3.39 and
3.78), and one acetyl methyl group (δH 1.97). The 13C NMR
spectrum showed the resonances of 22 carbons, which were
classified into two carbonyl carbons at 175.5 and 173.0 ppm,
eleven olefinic and aromatic carbons from 159.5 to 124.6 ppm,
two heteroatom bonded methine carbons at 78.0 and 55.0 ppm,
two methine carbons (δC 24.9 and 23.2), two methylene carbons
(δC 37.0 and 17.7) and three methyl carbons (δC 22.8, 14.2 and
11.9) by HSQC spectra (Figures S4-S6).
Figure 3. Key ROESY (bold arrows) and coupling constants
(dotted arrows) of 1.
The absolute configuration of 1 was defined by their
electronic circular dichroism (ECD) spectra, followed by
advanced Marfey’s analysis of acid hydrolysate derived from
desulfurization of
1
with Raney-nickel. The absolute
configuration of 1 at C-1, C-2 and C-3 was deduced by ECD
spectra10 in comparison with their calculated spectra11 (Figure
S2). Conformational searches were performed the Monte Carlo
algorithm implemented in Spartan’14 using the Merck molecular
force field (MMFF), optimized by semiempirical PM3
calculations in Gaussian 0912, then further refined by density
Figure 2. Key correlations observed in COSY (bold lines),
HMBC (arrows) and ROESY (dotted arrows) spectra of 1.

3
functional theory (DFT) at the CAM-B3LYP/TZVP level, which
yielded additional relevant conformers. Structures of resulting
calculated conformers were also supported by our ROESY
analyses and expected NMR calculation (Table S2 & 3). As a
result of the comparison between the experimental and calculated
ECD spectra, the absolute configuration of 1 was elucidated to be
1S,2R,3S. Combination of Raney-nickel desulfurization and
advanced Marfey’s method were performed in order to determine
the absolute configuration of N-acetyl-cysteine moiety in 1. After
1 was subjected to desulfurization with Raney-nickel in ethanol,
nickel was removed by filtration through a pad of celite.
Following alanine generated from the reacted solution using acid
hydrolysis, and the advanced Marfey’s procedure led to the
assignment of the absolute configuration of the alanine as S form
(Figure S3). Finally, the absolute configuration of 1 was
elucidated to be 1S,2R,3S,2’S.
Supplementary Material
Supplementary material that may be helpful in the review
process should be prepared and provided as a separate electronic
file. That file can then be transformed into PDF format and
submitted along with the manuscript and graphic files to the
appropriate editorial office.
References and notes
1.
2.
Hata, T.; Sano, Y.; Ohki, N.; Yokoyama, Y.; Matsumae, A., Ito, S.
J. Antibiot. 1953, 6, 87-89.
Burg, R. W.; Miller, B. M.; Baker, E. E.; Birnbaum, J.; Currie, S.
A.; Hartman, R.; Kong, Y. L.; Monaghan, R. L.; Olson, G.; Putter,
I.; Tunac, J. B.; Wallick, H.; Stapley, E. O.; Oiwa, R.; Ōmura, S.
Antimicrob. Agents Chemother. 1979, 15, 361-367.
Nakashima, T.: Iwatsuki, M.; Ochiai, J.; Kamiya, Y.; Nagai, K.;
Matsumoto, A.; Ishiyama, A.; Otoguro, K.; Shiomi, K.;
Takahashi, Y.; Ōmura, S. J. Antibiot. 2014, 67, 253-260. (b)
Nakashima, T.; Kamiya, Y.; Iwatsuki, M.; Takahashi, Y.; Ōmura,
S. J. Antibiot. 2014, 67, 533-539. (c) Nakashima, T.; Kamiya, Y.;
Iwatsuki, M.; Sato, N.; Takahashi, Y.; Ōmura, S. J. Antibiot. 2015,
68, 220-222.
3.
Iminimycin B (1) was tested for antibacterial and cytotoxic
activities. Compound 1 showed weak antibacterial activity
against Xanthomonas campestris pv. oryzae KB-88 (9 mm, 100
μg/disk). No significant inhibitory activities against HeLa S3 and
Jurkat cells were observed at 100 μM under the conditions tested.
4.
5.
6.
Nakashima, T.; Boonsnongcheep, P.; Kimura, T.; Iwatsuki, M.;
Sato, N.; Nonaka, K.; Prathanturarug, S.; Takahashi, Y.; Ōmura,
S. J. Biosci. Bioeng. 2015, 120, 596-600.
To our knowledge, there is no available information regarding
actinomycete secondary metabolites with a pyridinium ring
except for one compound, 1-(10-Aminodecyl) pyridinium, from a
marine actinomycete, Amycolatopsis alba.13 The actinomycete
producer of 1, which is a rare compound with pyridinium ring,
was S. griseus that isolated in 1972 and preserved for over 40
years. Our PC screening system led to the discovery of new
compounds, even from widely studied actinomycete species such
as S. griseus. Since 1 has a N-acetyl-cysteine moiety, which was
revealed to originate from mycothiol that involved in the
bacterial detoxification system,14 compound 1 may be derived for
inactivation of iminimycin A with antibacterial and cytotoxic
activities.
Nakashima, T.; Miyano, R.; Iwatsuki, M.; Shirahata, T.; Kimura,
T.; Asami, Y.; Kobayashi, Y.; Shiomi, K.; Petersson, G. A.;
Takahashi, Y.; Ōmura, S. J. Antibiot. 2016 (in press).
Carroll, A. R.; Arumugan, G.; Quinn, R. J.; Redburn, J.; Guymer,
G.; Grimshaw, P. J. Org. Chem. 2005, 70, 1889-1892. (b) Daly, J.
W.; Brown, G. B.; Mensah-Dwumah, M.; Myers, C. W. Toxicon
1978, 16, 163-188. (c) Jones, T. H.; Torres, J. A.; Spande, T. F.;
Garraffo, H. M.; Blum, M. S.; Snelling, R. R. J. Chem. Ecol.
1996, 22, 1221-1236.
Freer, A. A.; Gardner, D.; Greatbanks, D.; Polyser, J. P.; Sim, G.
A. J. Chem. Soc. Chem. Commun. 1982, 20, 1160–1162. (b)
Izumikawa, M.; Hosoya, T.; Takagi, M.; Shin-ya, K. J. Antibiot.
2012, 65, 41-43.
7.
8.
9.
Gomi, S.; Ikeda, D.; Nakamura, H.; Naganawa, H.; Yamashita, F.;
Hotta, K.; Kondo, S.; Okami, Y.; Umezawa, H.; Iitaka Y. J.
Antibiot. 1984, 37, 1491-1494.
Acknowledgments
Yoshida, W. Y.; Scheuer P. J. Heterocycles 1998, 47, 1023– 1027.
We are grateful to Drs Kenichiro Nagai and Noriko Sato,
School of Pharmacy, Kitasato University, for measurement of
MS and NMR spectra. This study was supported by funds from
the Institute for Fermentation, Osaka (IFO), Japan.
10. Stonard, R. J.; Trainor, D. A.; Nakatani, M.; Nakanishi, K. J. Am.
Chem. Soc. 1983, 105, 130-131. (b) Li, X. -C.; Ferreira, D.; Ding,
Y. Curr. Org. Chem. 2010, 14, 1678– 1697.
11. Nugroho, A. E.; Morita, H. J. Nat. Med. 2014, 68, 1-10.
12. Frisch, M. J. et al. Gaussian 09, Revision D.01 (Gaussian,
Wallingford, CT, 2009).
13. Dasari, V. R.; Muthyala, M. K.; Nikku, M. Y.; Donthireddy, S. R.
Microbiol. Res. 2012, 167, 346-351.
14. Ikeda, H.; Shin-Ya, K.; Nagamitsu, T.; Tomoda, H. J. Ind.
Microbiol. Biotechnol. 2016, 43, 325-342.

4
Tetrahedron
Highlights
･ Iminimycin B was discovered from Streptomyces
griseus OS-3601 by a physicochemical screening.
･ Structure of iminimycin B was an unique novel
pyridinium alkaloid.
･ Absolute configuration of iminimycin B was
determined by NMR, eECD analysis and Marfey’s
method.