Simamycin (5′-O-geranyluridine): A new prenylated nucleoside from Streptomyces sp. A

Article abstract of DOI:10.1038/ja.2016.163

A new nucleoside modified by prenylation, simamycin (1), was isolated from the culture broth of a soil-derived Streptomyces sp. Its structure was determined by spectroscopic analysis and chemical synthesis as 5′-O-geranyluridine. Compound 1 induced differentiation of preadipocytes into matured adipocytes.

Full text of DOI:10.1038/ja.2016.163

The Journal of Antibiotics (2017), 1–4
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
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&
ORIGINAL ARTICLE
Simamycin (5′-O-geranyluridine): a new prenylated
nucleoside from Streptomyces sp.
Yasuhiro Igarashi1, Takayuki Kyoso1, Youngju Kim1 and Tsutomu Oikawa2
A new nucleoside modified by prenylation, simamycin (1), was isolated from the culture broth of a soil-derived Streptomyces sp.
Its structure was determined by spectroscopic analysis and chemical synthesis as 5′-O-geranyluridine. Compound 1 induced
differentiation of preadipocytes into matured adipocytes.
The Journal of Antibiotics advance online publication, 18 January 2017; doi:10.1038/ja.2016.163
INTRODUCTION
Despite the substantial amounts of studies on its secondary metabo-
Compound 1 was obtained as optically active colorless needles that
gave an [M+H]+ pseudomolecular ion at m/z 381.2027 appropriate for
lites, Streptomyces is still believed to be the most prolific source of the molecular formula of C19H29N2O6 (7 degrees of unsaturation).
This molecular formula was corroborated by 1H and 13C NMR
novel bioactive compounds. In fact, up to 40% of known microbial
spectral data (Table 1). The IR absorptions at 1694 and 1664 cm− 1
suggested the presence of carbonyl functionalities. One- and 2D NMR
data of 1 revealed the presence of 19 carbons, which were assigned to
four quaternary sp2 carbons including two oxygenated carbons, four
proton-bearing sp2 carbons, six oxygenated sp3 carbons, two aliphatic
methylenes and three methyls.
metabolites are derived from this group.1 However, it is noteworthy
that the number of metabolites isolated is far below the number of
gene clusters for secondary metabolite biosynthesis present in the
genome of Streptomyces species.2,3 In our screening for structurally
unique or rare secondary metabolites from Streptomyces, we isolated
plant hormone-like spiroacetals of polyketide origin,4 a linear polyke-
tide δ-lactone with antimetastatic property,5 macrocyclic polyketides
with a biosynthetically unprecedented heterocyclic ring structure6
and methylphenylalanine derivatives with adipocyte differentiation
promoting activity.7 Further attempts to obtain new bioactive
compounds from Streptomyces, a soil-derived strain TP-A0872, was
chosen for chemical investigation, which led to the isolation of a new
geranylated nucleoside, simamycin (1; Figure 1).
From the 1H-1H COSY spectrum, five proton-bearing fragments
were established: H-15/H-16/H-17, H-12/H-13, H-10/H-11, H-7/H-8
and H-5/H-6. The first and the second fragments were joined at C-14
on the basis of HMBC correlations from the methyl protons H-20 to
C-13, C-14 and C-15. This fragment was expanded to include a three-
carbon fragment C-19/C-18/C-21 on the basis of HMBC correlations
from H-19 and H-21 to C-17 and C-18, and to one another, providing
a geranyl moiety. The third and the fourth fragments were connected
at C-9 by HMBC correlations from H-8 to C-9 and C-10, H-9 to C-8
and C-11, and H-10 and H-11 to C-9, establishing connectivity from
C-7 to C-11. Furthermore, HMBC correlations from H-7 to C-10 and
H-10 to C-7 connected C-7 and C-10 via an ether linkage, to confirm
a pentofuranosyl residue. The last fragment H-5/H-6 was extended to
include two carbonyl carbons C-2 and C-4, and the latter carbon was
connected to C-5 by HMBC correlations from H-5 and H-6 to C-4.
As the molecular formula suggested the presence of two nitrogen
atoms in 1, analysis of 1H-15N long-range correlations became
RESULTS AND DISCUSSION
The producing strain TP-A0872 was isolated from a soil sample
collected in Kochi, Japan, and identified as a member of the genus
Streptomyces on the basis of 16S rRNA gene sequence analysis. The
whole culture broth of strain TP-A0872 cultured in A-3M liquid
medium was extracted with 1-butanol. HPLC/UV analysis of the
extract and dereplication using our in-house metabolite database
suggested the presence of an unknown peak showing a UV absorption
band at 263 nm. HPLC/UV-guided purification from the extract led to
the isolation of a new geranylated nucleoside, 1. Subsequent biological
evaluation elucidated that 1 induces the differentiation of preadipo-
cytes into matured adipocytes. We herein describe the isolation,
structure determination and biological activity of 1.
1
necessary to locate their positions. In the H-15N HMBC spectrum,
both H-5 and H-6 showed a cross-peak to a nitrogen at 156.3 p.p.m.
and H-5 to a nitrogen at 155.7 p.p.m. These correlation data, together
with 13C and 15N chemical shifts, established this chromophore as
pyrimidine-2,4(1H,3H)-dione, namely uracil. Finally, three
1Department of Biotechnology, Biotechnology Research Center, Toyama Prefectural University, Toyama, Japan and 2Department of Nutritional Biochemistry, School of Nutrition
and Dietetics, Kanagawa University of Human Services, Kanagawa, Japan
Correspondence: Professor Y Igarashi, Department of Biotechnology, Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398,
Japan.
E-mail: yas@pu-toyama.ac.jp
This paper is dedicated to Professor Dr Satoshi Ōmura for his Nobel Prize in Physiology or Medicine 2015.
Received 21 October 2016; revised 1 December 2016; accepted 6 December 2016

Geranylated nucleoside from Streptomyces
Y Igarashi et al
2
Figure 1 Structure of simamycin (1).
Table 1 1H and 13C NMR data for 1 in CDCl3
Figure 2 COSY and key HMBC correlations for 1.
b
Position
δH mult (J in Hz)a
δC
HMBCc
2
151.6
164.2
102.2
141.0
90.5
4
5
5.66 d (8.0)
8.00 d (8.0)
5.92 d (3.0)
4.23
4, 6
2, 4, 5, 7
2, 6, 8, 10
9, 10
6
7
8
75.8
9
4.21
84.4
8, 11
10
11
4.22
70.7
7
3.76 d (10.5)
3.60 d (10.5)
4.04
68.7
9, 10, 12
9, 10, 12
11, 13, 14
12, 15, 20
Figure 3 Key NOESY correlations for 2. A full color version of this figure is
available at The Journal of Antibiotics journal online.
12
13
14
15
16
17
18
19
20
21
67.9
120.2
141.3
39.8
5.29 t (6.5)
in HCl to afford 1 (Scheme 1). Optical rotation of the synthetic 1 was
[α]D24 +16 (c 0.1, CHCl3), which was in good accordance with that of
24
2.05
2.10
13, 14, 17
17
natural 1, [α]D +13 (c 0.1, CHCl3). The absolute configuration of 1
26.6
was thus determined as depicted in Figure 1.
5.08 t (6.5)
123.9
131.5
25.9
16, 19, 21
Compound
1 was not active against Staphylococcus aureus,
Escherichia coli and Candida albicans in the antimicrobial assay
(MIC 450 μg ml− 1). It was also inactive in cytotoxicity assay
(IC50 450 μM against MCF7 human breast cancer cells). However,
1 was found to induce differentiation of murine ST-13 preadipocyte
cells into matured adipocyte cells.10 Maturation of adipocytes leads to
the secretion of adiponectin, which downregulates the blood glucose
level.11 About 50% of preadipocytes were differentiated to adipocytes
with accumulation of lipid droplets by the treatment with 40 μM of 1
for 11 days (Figure 4). JBIR-68 in which the double bond of uridine of
1 is hydrogenated was reported to inhibit influenza proliferation12 but
we had no access to this assay.
1.73 s
1.68 s
1.60 s
17, 18, 21
13, 14, 15
17, 18, 19
16.6
17.9
aRecorded at 500 MHz.
bRecorded at 125 MHz.
cHMBC correlations are from proton(s) stated to the indicated carbon.
substructures were joined by analysis of HMBC data. HMBC correla-
tions from H-11 and H-12 to one another connected the geranyl
group at C-11 of the pentose unit via an ether linkage, whereas the
uracil moiety was connected N-1 to the anomeric carbon (C-7) of the
pentose unit by HMBC correlations from H-7 to C-2 and C-6, and
H-6 to C-7. Thus, the gross structure of 1 was determined as shown in
Figure 2.
CONCLUSION
In summary, we found 1, a new nucleoside modified by O-prenylation
As the signal overlapping of H-8, H-9 and H-10 in the 1H
NMR spectrum did not allow the stereochemical assignment of
the pentose unit by NOE and J-based analysis, 1 was derivatized to
bis-p-bromobenzoate (2) by the treatment with p-bromobenzoyl
chloride in pyridine. The above mentioned proton signals were
separated each other in the 1H NMR spectrum of 2. NOESY
correlations were detected for H-8/H-9, H-7/H-10, H-2/H-8 and
H-9/H-11 (Figure 3). These correlation data allowed the assignment of
the pentose unit to possess a ribose configuration, thereby establishing
1 as 5′-O-geranyluridine.
To determine the absolute configuration of the ribose unit, 1 was
synthesized starting from commercially available natural (+)-enantio-
mer of uridine in three steps. The cis-diol of uridine was protected
as dimethylacetonide,8 which was then O-alkylated using geranyl
bromide in the presence of NaH to give 4.9 Geranylation at the
5-position of ribose was confirmed by an HMBC correlation from
H-12 to C-11. Additional evidence for the alkylation site was the
HMBC correlations from 3-NH (δH 9.58) to C-4 (δC 163.6) and C-5
with
a geranyl group from a soil-derived Streptomyces by
UV-spectroscopic screening. Prenylation of nucleosides is very rare
in nature. To date, JBIR-68 (Takagi et al.12) and farnesides A and B13
are known as a secondary metabolite of Streptomyces. JBIR-68 is a
dihydrouridine derivative in which the hydroxy group at five-position
of ribose is modified with a geranyl group. Farnesides are also
dihydrouridine derivatives but an oxygenated farnesyl group is
connected to the hydroxy group at five-position of the ribose moiety.
EXPERIMENTAL PROCEDURE
General experimental procudures
Optical rotations were measured using a Jasco P-1030 polarimeter (JASCO
Corporation, Tokyo, Japan). UV spectra were recorded on a Hitachi U-3210
spectrophotometer (Hitachi-High-Technologies Co., Tokyo, Japan). IR spectra
were recorded on a Shimadzu FT-IR-300 spectrophotometer (Shimadzu Corp.,
Kyoto, Japan). NMR spectra were obtained on a Bruker AVANCE II 500
spectrometer (Bruker Biospin K. K., Yokohama, Japan). HR-FAB-MS and HR-
ESI-MS were measured on a JEOL JMS-HX110A spectrometer (JEOL Ltd.,
Tokyo, Japan) and a Bruker micrOTOF (Bruker Daltonics K. K., Yokohama,
(δC 102.0). The acetonide protective group was removed by treating 4 Japan), respectively.
The Journal of Antibiotics

Geranylated nucleoside from Streptomyces
Y Igarashi et al
3
Scheme 1 Synthesis of 1. Reagents: (a) 2,2-dimethoxypropane, TsOH; (b) geranylbromide, NaH; (c) dil HCl.
solution was extracted with EtOAc. The organic layer was concentrated to give
16
12
8
1 (8.8 mg) as a colorless solid.
Simamycin (5-O0-geranyluridine, 1). Colorless needles; mp 95–96 °C; [α]D
24
+13 (c 0.10, CHCl3); UV λmax (log ε) 263 nm (3.90); IR (KBr) νmax 3377, 1694,
1664 cm− 1; For 1H and 13C NMR data, see Table 1. 15N NMR (CDCl3) δ 156.3
(N-1), 155.7 (N-3) (chemical shifts were indirectly determined from the
projection of 1H-15N HMBC spectrum); HR-FABMS [M+H]+ m/z 381.2027
(calcd for C19H30N2O6, 381.2025).
Bis-p-bromobenzoate of 1 (2)
4
To a solution of 1 (3.0 mg, 7.9 μmol) in dry CH2Cl2 (1.2 ml) and dry pyridine
(0.6 ml) were added N,N-dimethyl-4-aminopyridine (a trace amount) and p-
bromobenzoyl chloride (3.5 mg, 16 μmol) at 0 ~ 5 °C. After stirring for 20 h at
room temperature, the reaction mixture was diluted with ice water and
extracted with EtOAc. The organic layer was successively washed with sat.
CuSO4 solution, sat. NaHCO3 solution and brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (hexane-EtOAc, 10:1~ 1:1) to give 2 (1.0 mg,
0
0
1.25 2.5
5
10 20 40 ROS
Simamycin (µM)
(ROS: 0.02 µM rosiglitazone)
1
Figure 4 Adipocyte differentiation induced by 1. A full color version of this
figure is available at The Journal of Antibiotics journal online.
17% yield) as a light yellow solid; H NMR (500 MHz, CDCl3) δ 8.16 (1H, s),
5.76 (1H, d, J = 8.2 Hz, H-5), 7.99 (1H, d, J = 8.2 Hz, H-6), 6.53 (1H, d,
J = 7.7 Hz, H-7), 5.62 (1H, dd, J = 7.7, 5.6 Hz, H-8), 5.73 (1H, m, H-9), 4.48
(1H, br.s, H-10), 3.81 (1H, dd, J = 9.4, 1.7 Hz, H-11), 3.72 (1H, dd, J = 9.4,
1.7 Hz, H-11), 4.18 (1H, dd, J = 11.6, 6.8 Hz, H-12), 4.13 (1H, dd, J = 11.6,
7.2 Hz, H-12), 5.37 (1H, t, J = 6.5 Hz, H-13), 2.31 (2H, m, H-15), 2.09 (2H, m,
H-16), 5.02 (1H, t, J = 6.6 Hz, H-17), 1.67 (3H, s, H-19), 1.73 (3H, s, H-20),
1.57 (3H, s, H-21), 7.84 (2H, d, J = 8.5 Hz), 7.46 (2H, d, J = 8.5 Hz), 7.73 (2H,
d, J =8.5 Hz), 7.58 (2H, d, J = 8.5 Hz); 13C NMR (125 MHz, CDCl3) δ 150.3
(C-2), 162.4 (C-4), 103.3 (C-5), 140.0 (C-6), 85.9 (C-7), 74.2 (C-8), 73.5 (C-9),
83.1 (C-10), 69.2 (C-11), 67.9 (C-12), 119.4 (C-13), 142.3 (C-14), 39.6 (C-15),
26.4 (C-16), 123.6 (C-17), 131.98 (C-18), 25.7 (C-19), 16.5 (C-20), 17.7
(C-21), 127.3, 127.7, 129.07, 129.13, 131.2, 131.3, 132.0, 132.1, 164.5, 164.8;
HR-FABMS [M+Na]+ m/z 767.0580 (calcd for C33H34N2O8Br2Na, 756.0579).
Microorganism
The actinomycete, Streptomyces sp. TP-A0872, was isolated from a soil sample
collected in Kochi, Japan. The isolated strain was identified as Streptomyces on
the basis of 99.9% similarity in the 16S rRNA gene sequence (1463 nucleotides;
DDBJ accession number LC192168) to Streptomyces xanthophaeus NRRL
B-5414T (accession number JOFT01000080).
Fermentation
Strain TP-A0872 cultured on a slant agar medium was inoculated into 500 ml
K-1 flasks, each containing 100 ml of the seed medium consisting of soluble
starch 1%, glucose 0.5%, NZ-case 0.3%, yeast extract (Kyokuto Pharmaceutical
Industrial, Co., Ltd, Tokyo, Japan) 0.2%, Tryptone (Difco Laboratories, Sparks,
MD, USA) 0.5%, K2HPO4 0.1%, MgSO4·7H2O 0.05% and CaCO3 0.3% (pH
7.0). The flasks were cultivated on a rotary shaker (200 r.p.m.) at 30 °C for
4 days. The seed culture (3 ml) was transferred into 500 ml K-1 flasks each
containing 100 ml of the production medium consisting of glucose 0.5%,
glycerol 2%, soluble starch 2%, Pharmamedia (Traders Protein) 1.5%, yeast
extract 0.3% and Diaion HP-20 (Mitsubishi Chemical Co., Yokohama, Japan)
1%. The pH of the medium was adjusted to 7.0 before sterilization. The
inoculated flasks were cultured on a rotary shaker (200 r.p.m.) at 30 °C for
7 days.
5-O-Geranyl-2,3-O-isopropylidene-1-uracil-β-D-ribofuranoside (4)
To a solution of uridine (700 mg, 2.87 mmol) in dry N,N-dimethylformamide
(70 ml) were added 2,2-dimethoxypropane (7 ml, 57 mmol) and p-toluene-
sulfonic acid monohydrate (70 mg, 0.37 mmol) at room temperature. After
stirring for 18 h, the reaction was quenched by adding saturated NaHCO3
solution to the reaction mixture until the pH became 7.0. The reaction mixture
was diluted with distilled water and loaded onto an HP-20 column and the
column was washed with distilled water. The column was then eluted with
MeOH and the eluent was concentrated under reduced pressure to give 2,3-O-
isopropylidene-1-uracil-β-D-ribofuranoside (3, 500 mg, 61% yield). Spectro-
scopic data were in good agreement with reported values.9
Extraction and isolation
The whole culture broth of strain TP-A0872 (3 l) was extracted with 1-butanol
(100 ml per flasks) on a rotary shaker (200 r.p.m.) for 1 h. The mixture was
centrifuged at 6000 r.p.m. for 10 min and the organic layer was separated from
the aqueous layer containing the mycelium. Evaporation of the solvent in vacuo
provided 5.23 g of extract, which was subjected to silica gel column
chromatography with a step gradient of CHCl3-MeOH (16:1, 8:1, 4:1, 2:1,
1:1 and 0:1 v/v). Concentration of fraction 2 (CHCl3-MeOH =8:1) provided
1.1 g of brown powders, which was further purified by HPLC separation with a
To a suspension of 3 (120 mg, 0.42 mmol) in a mixture of dry benzene
(1.45 ml) and dry 1,4-dioxane (0.45 ml) was added NaH (40% purity, 60 mg,
2.5 mmol) under argon atmosphere. The mixture was heated at 80~ 100 °C for
30 min with stirring. After cooling to the ambient temperature, geranyl
bromide (0.17 ml, 0.89 mmol) was added and the reaction mixture was heated
at 90~ 95 °C for 3 h. The reaction was quenched by adding water at 0 ~ 5 °C
and the mixture was extracted with EtOAc. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was applied
gradient of MeCN-0.15% KH2PO4 buffer (pH 3.5) (0–6 min: 40%; 6–20 min: to silica gel column chromatography (n-hexane:EtOAc = 16:1~ 1.1) to give
40–50%; 15 ml min−1). Compound 1 was eluted at 17.8 min. Fraction was 4 (39 mg, 22% yield): 1H NMR (500 MHz, CDCl3): δ 9.58 (1H, s), 7.68
pooled and concentrated under reduced pressure and the remaining aqueous (1H, d, J = 8.0 Hz), 5.95 (1H, d, J = 3.0 Hz), 5.67 (1H, d, J = 8.0 Hz), 5.29
The Journal of Antibiotics

Geranylated nucleoside from Streptomyces
Y Igarashi et al
4
(1H, t, J = 5.5 Hz), 5.06 (1H, t, J = 5.5 Hz), 4.79 (1H, dd, J = 6.0, 3.0 z), 4.78
(1H, dd, J = 4.0, 2.5 Hz), 4.39 (1H, dd, J = 3.5, 3.0 Hz), 4.03 (1H, d, J = 7.0 Hz),
3.69 (1H, dd, J = 8.0, 2.5 Hz), 3.60 (1H, dd, J = 8.0, 3.5 Hz), 2.09 (2H, m), 2.05
(2H, m), 1.68 (3H, s), 1.66 (3H, s), 1.06 (3H, s), 1.59 (3H, s), 1.36 (3H, s); 13C
NMR (125 MHz, CDCl3) δ 16.5, 17.7, 25.3, 25.7, 26.3, 27.2, 39.6, 67.8, 69.8,
81.8, 85.1, 85.6, 92.6, 102.0, 114.1, 119.9, 123.7, 131.8, 141.1, 141.3, 150.3,
163.6; HR-ESITOFMS [M+Na]+ 443.2153 (calcd for C22H32N2O6Na,
443.2153).
1
2
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Synthetic 1
A stirred solution of 4 (9.4 mg, 22 μmol) in 95% EtOH (3 ml) and 0.1 M HCl
(1 ml) was heated at 70~ 80 °C for 4 h. After cooling to the ambient
temperature, 0.1 M NaOH solution was added to adjust the pH to 7.0. The
reaction mixture was then extracted with EtOAc and the organic layer was
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The
residue was purified by preparative HPLC in the same manner described for
natural simamycin to yield 6.3 mg of 1 (74% yield): colorless solid; [α]24D +16
(c 0.10, CHCl3); 1H and 13C NMR data were in good agreement with those for
natural 1; HR-ESITOFMS [M+Na]+ 403.1840 (calcd for C19H28N2O6Na,
403.1839).
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
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The Journal of Antibiotics