Isolation and structural determination of a new macrolide, makinolide, from the newly isolated Streptomyces sp. MK-30

Full text of DOI:10.1038/ja.2012.24

The Journal of Antibiotics (2012) 65, 331–334
2012 Japan Antibiotics Research Association All rights reserved 0021-8820/12
www.nature.com/ja
&
NOTE
Isolation and structural determination of a new
macrolide, makinolide, from the newly isolated
Streptomyces sp. MK-30
1
,2
3
1
1
3
Shinya Kodani , Ayako Murao , Michitaka Hidaki , Kanna Sato and Naoto Ogawa
The Journal of Antibiotics (2012) 65, 331–334; doi:10.1038/ja.2012.24; published online 11 April 2012
Keywords: anti-yeast compound; macrolide; NMR spectrum; Streptomyces
Macrolides are a group of organic compounds that possess a related to Streptomyces sahachiroi, with a high similarity of 99%. The
macrolide ring (usually 14-, 15- or 16-membered) as the carbon 16S rDNA sequence of Streptomyces sp. MK-30 was deposited in the
skeleton. Most of the isolated macrolides are structurally diverse DDBJ database under the accession number AB691771.
antibiotics such as erythromycin1,2 and are produced by
To isolate the anti-yeast compounds, the cultivation of the strain
microorganisms such as the soil-dwelling bacterium Streptomyces MK-30 was performed with 1 l of ISP2 agar media. After 7 days of
3
via polyketide biosynthesis. They exhibit various biological activities, cultivation, the spent agar culture media were extracted with an equal
including antitumor, antifungal, antiparasitic and immuno- volume of acetone. After concentration, the acetone extract was
4
,5
suppressant activities. Among the macrolides, bafilomycins and subjected to open column chromatography using the synthetic
setamycin6,7 are 16-membered macrolides that are reported to exhibit hydrophobic resin CHPꢀ20P and eluted with 20% MeOH, 60%
specific membrane ATPase inhibitory activity.8 In the course of MeOH and then MeOH. As the MeOH fraction showed potent
screening for anti-yeast compounds, we isolated a new bafilomycin activity, the MeOH fraction was repeatedly subjected to preparatory
analog named makinolide (1 in Figure 1a), along with the known HPLC to yield the new macrolide makinolide (1, 3.3 mg) and the
9
,10
macrolide JBIR ꢀ100 (2 in Figure 1a)
from newly isolated strain, known macrolide JBIRꢀ100 (2, 2.0 mg).
Streptomyces sp. MK-30. Herein, we describe the isolation and
structural determination of makinolide (1).
Makinolide was isolated as a white amorphous powder. HR–ESI–
þ
MS analysis of 1 gave an [M þ Na] ion at m/z 822.44272, consistent
The new bacterial strain MK-30 was isolated from the soil of a tea with the molecular formula C H NO (calculated [Mþ Na]þ ion
4
4
65
12
field (Makinohara, Shizuoka, Japan) along with 32 other bacterial at m/z 822.44044). To obtain further information on the chemical
1
strains using ISP2 agar medium. With the aim of screening for anti- structure, NMR experiments including H, HMQC, HMBC, DQF-
yeast compounds, these strains were cultured with ISP2 agar media for COSY and TOCSY analyses of 1 were performed in acetone ꢀd6
7
days. The spent culture media were extracted with acetone. Then, (Supplementary Figures S2ꢀS7).
each acetone extract was subjected to anti-yeast activity screening
against Saccharomyces cerevisiae (NBRC2376), and the strain MK-30 in the HMQC spectrum (Table 1). As shown by the bold line in
was found to produce a significant amount of anti-yeast substances. Figure 1b, the analysis of the TOCSYand DQF-COSY spectra revealed
To identify the genetic position of the MK-30 strain, sequencing proton–proton spin connections in five partial structures: carbon
The direct bonds between each proton and carbon were established
0
0
analysis was performed on the 16S ribosomal RNA (rRNA) coding skeletons connecting C5 to C9, C11 to C18, C20 to C25, C2 to C3
0
0
gene. Using a typical polymerase chain reaction (PCR) method with and C7 to C8 . The HMBC correlations between the protons of the
universal primers for the bacterial 16S rRNA gene, the nearly complete 2-Me group and C1, C2 and C3, and between the protons of the 4-Me
length of the 16S rRNA gene was amplified. The sequence analysis was group and C3,C4 and C5, indicated a 2, 4-dimethyl dienone structure.
accomplished using an automated DNA sequencer with eight universal The HMBC correlations between the protons of the 10-Me group and
primers. From the obtained sequence, a phylogenetic tree was C9, C10 and C11 indicated a connection between the (C5 to C9) and
constructed using the multiple-alignment program ClustalX. It was (C11 to C18) partial structures. The HMBC correlations between the
determined that the genetic position of MK-30 is located in the genus protons of the 18-Me group, H18, H20 and C19 indicated a
Streptomyces (Supplementary Figure S1). The strain MK-30 is closely hemiacetal structure, which was also confirmed by the characteristic
1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Suruga-ku, Shizuoka, Japan; 2Graduate School of Science and Technology, Shizuoka
University, Suruga-ku, Shizuoka, Japan and 3Department of Biological and Environmental Science, Faculty of Agriculture, Shizuoka University, Suruga-ku, Shizuoka, Japan.
Correspondence: Dr Shinya Kodani, Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
E-mail: askodan@ipc.shizuoka.ac.jp
Received 1 September 2011; revised 28 February 2012; accepted 6 March 2012; published online 11 April 2012

Structural determination of makinolide
S Kodani et al
3
32
Figure 1 (a) Chemical structure of makinolide (1) and JBIR-100 (2). (b) Selected NMR correlations of 1.
chemical shift value for C19 (dc 99.4). The HMBC correlation PCR amplification, sequencing and phylogenetic analysis of 16S
between H23 and C19 was not observed; however, a comparison of rRNA genes
the chemical shift values with the related compound JBIRꢀ100 The extraction of all DNA from the cells of the strain MK-30 was performed
according to the literature procedure.13 The 16S rRNA-encoding sequence was
confirmed the cyclic hemiacetal moiety. The presence of the N-(3-
amplified from the total DNA by the PCR method with two sets of universal
hydroxy-2-cyclopentenone-2-yl)-fumaric acid monoamide moiety
0 0 0
primer pairs: 9F (5 -GAGTTTGATCCTGGCTCAG-3 ) and 926R (5 -CCG
0 0
(
C1 –C7 ) was confirmed by the chemical shifts observed in the
0
0
0
0
TCAATTCCTTTGAGTTT-3 ); and 686F (5 -TAGCGGTGAAATGCGTAGA-3 )
0
HMQC spectrum and the molecular formula determined by the HR-
ESI-MS analysis. Although no HMBC correlations were observed
and 1510R (5 -GGCTACCTTGTTACGA-3 ). For PCR, the reaction mixture
was prepared by adding 1.0 ml of the total DNA of the MK-30 strain (100ng),
0
0
0
between H7 and H8 , the similarity of the chemical shift values for
0
1.0 ml of Pfu DNA polymerase (Bioneer, Daejeon, South Korea), 5.0ml of X10
the 7 and 8 positions to the values for these positions in the related
reaction buffer (Bioneer), 4ml of 2.5 mM dNTP mix solution (Bioneer) and
compound R17650211 support the structural assignment. Previously, 31 ml of distilled water into the PCR reaction tube. PCR amplification was
the acid hydrolysis of bafilomycin B1 was reported to form N-(3- carried out using a thermal cycler using the following program: initial
hydroxy-2-cyclopentenone-2-yl)-fumaric acid monoamide.12 To denaturation for 10min at 941C, followed by 34 cycles consisting of
denaturation for 40 s at 941C, annealing for 60s at 551C and DNA
synthesis for 1 min at 721C. A final extension of 5 min at 721C was
included at the end of the 34 cycles. The PCR product was purified with a
Qiagen PCR PURE kit (Qiagen, Venlo, Netherlands) following the
manufacturer’s instructions. The reactions for sequencing were performed
using a Beckman DTCS-Quick Start Kit following the manufacturer’s
confirm the partial structure containing the N-(3-hydroxy-2-
0
0
cyclopentenone-2-yl)-fumaric acid monoamide moiety (C1 –C7 ),
mild acid hydrolysis of 1 was accomplished following the procedure
described in the previous report.12 The chemical shifts in the 1
H
NMR spectrum (in MeOH ꢀd ) of the obtained compound were
4
identical to those observed in the spectrum of N ꢀ(3 ꢀhydroxy ꢀ2-
0
instructions. Four primers were used for the reaction: 339F (5 -CTCC
0
cyclopentenone-2-yl)-fumaric acid monoamide derived from an
0
0
0
TACGGGTGAGTAACAC-3 ), 536R (5 -GTATTACCGCGG CTGCTG-3 ),
0
0
authentic sample of bafilomycin B1, which confirmed the partial 686F (5 -TAGCGGTGAAATGCGTAGA-3 ) and 1099F (5 -GCAACGAGCGC
0
0
0
structure of the C1 –C7 skeleton (Supplementary Figures S8 and S9). AACCC-3 ). The sequencing was performed with the capillary DNA sequencer
In the disc difussion assay, the sensitivity of various microorgan- CEQ8000XL (Beckman Coulter, Brea, CA, USA).
isms was tested against macrolides 1 and 2 following previously
4
reported procedures (Table 2). Among the bacteria, compounds 1 Isolation of the bacterial strains
and 2 showed inhibitory activities only against Micrococcus luteus at The soil samples were collected from a tea-field ground in Makinohara,
the doses of 15 and 50 mg per disc. Bafilomycins have been reported to Shizuoka, Japan. The soil samples were spread onto ISP2 agar medium. After
be specific inhibitors of the membrane ATPase of eucaryotic cells,8
and recently, 2 was confirmed to exhibit inhibitory activity against
4–5 days of incubation at 30 1C, the colonies that developed were isolated and
stored in a refrigerator at ꢀ801C. In total, 33 strains were collected and
cultured for anti-yeast compound screening. Briefly, the ISP agar culture
vacuolar-type ATPase.9 Considering the inhibitory mechanism of
(
acetone extract was evaporated and dissolved in dimethyl sulfoxide to adjust
25 ml) of each strain was extracted with an equal volume of acetone. Each
bafilomycins, the related compounds 1 and 2 were thought to be
active against yeast and fungi; however, among the six strains of yeast
and fungi that were tested, macrolides 1 and 2 exhibited inhibitory
activities against only S. cerevisiae and Mucor hiemalis (Table 2).
ꢀ1
the concentration to 10 mgml . The anti-yeast activity was evaluated by
determining the inhibitory zone that was caused by a 10-mg inoculation of the
acetone extract sample on a culture of the yeast S. cerevisiae using ISP2 agar
medium.
EXPERIMENTAL PROCEDURE
General Methods
NMR spectra were obtained using a JEOL ECA-600 in acetone-d6 or MeOH-d4
Isolation of makinolide
ISP2 agar medium (1l) was used for culturing Streptomyces sp. MK-30. After 7
(
using a JEOL JMS-T100LP mass spectrometer.
JEOL Ltd, Tokyo, Japan) at 27.01C. The ESI–MS spectrum was recorded days at 301C, an equal volume of acetone was added to the agar culture for
extraction. The acetone extract was concentrated to an aqueous suspension,
The Journal of Antibiotics

Structural determination of makinolide
S Kodani et al
3
33
Table 1 NMR chemical shift values of makinolide (1) in acetone–d6
Table 2 Antimicrobial activities of 1 and 2
Position
dC
dH (J in Hz)
Diameter of inhibition zone (mm)
1
2
174.0
121.3
13.5
147.9
134.0
15.4
148.4
38.0
17.5
81.0
39.0
22.4
43.0
145.5
20.2
125.0
134.0
127.0
84.0
56.0
77.0
38.0
12.5
72.0
42.8
24.0
99.4
40.5
76.0
38.2
22.6
75.2
28.5
21.7
14.8
164.9
130.2
135.0
166.9
ꢀ
1
2
2
3
4
4
5
6
6
7
8
8
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
4
5
6
7
8
9
-CH3
-CH3
-CH3
-CH3
2.02 (s)
7.25 (s)
Microorganisms
15 mg
50mg
15 mg
50mg
oBateria4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
13
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
20
ꢀ
1.96 (s)
Escherichia coli (NBRC 1002203)
Pseudomonas aeruginosa (NBRC 12689)
Serratia marcescens (NBRC 1002204)
Vibrio harveyi (NBRC 15634)
Bacillus subtilis (NBRC13719)
Staphylococcus aureus (NBRC 1009110)
Micrococcus luteus (NBRC3333)
Streptomyces antibioticus (NBRC 3117)
oYeast4
Saccharomyces cerevisiae (NBRC2376)
Schizosaccharomyces pombe (NBRC 0340)
Kloeckera apiculata (NBRC 0154)
oFungi4
6.04 (d, 9.0)
2.56 (m)
ꢀ
ꢀ
ꢀ
ꢀ
1.03 (d, 6.9 )
3.29 (m)
ꢀ
ꢀ
ꢀ
ꢀ
1.89 (s)
ꢀ
ꢀ
0.90 (d, 6.9)
1.73 (m)
15
18
ꢀ
ꢀ
0
0-CH3
1.90 (s)
12
ꢀ
ꢀ
13
ꢀ
ꢀ
11
ꢀ
ꢀ
9
ꢀ
ꢀ
1
5.77 (d, 11.0)
6.67 (d, 15.1, 10.3)
5.12 (dd, 15.1, 9.6)
4.07 (m)
2
3
4
Aspergillus niger (NBRC 33023 )
Aspergillus oryzae (NBRC 4290)
Mucor hiemalis (NBRC 9405)
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
4-OCH3
3.20 (s)
ꢀ
11
ꢀ
12
5
4.91 (m)
15
18
6
2.08 (m)
6-CH3
0.83 (d, 6.8)
4.10 (m)
Paper discs (i.d. 6 mm for 15mg per disc and i.d. 8 mm for 50mg per disc) were used.
The minus ( ꢀ) means no inhibition.
7
8
1.80 (m)
4
.6 ꢁ 250 mm) using isocratic elution of 13% MeCN containing 0.05% TFA
8-CH3
0.96 (d, 7.6)
and a UV detector set at an absorbance of 220 nm to yield N-(3-hydroxy-2-
cyclopentenone-2-yl)-fumaric acid monoamide: HR ESI-MS showed an ion
9
0
1.26 (m), 2.31 (dd, 11.5, 4.8)
5.01 (m)
þ
peak at m/z 234.0383 [Mþ Na] calculated for C H O Na (D 0.55mmu).
9
9 5
1
1
0
The H-NMR signals (MeOH-d4) were detected at dH H2 : 7.18 (d, 15.1Hz,
0 0 0
2
1.60 (m)
1
H), H3 : 6.81 (d, 15.1Hz, 1 H), and H8 and H9 : 2.54 (s, 4 H).
2-CH3
0.84 (d, 6.9)
3
4
5
6
’
3.59 (dd, 10.3, 2.1)
1.91 (m)
Bacterial strains for antimicrobial assay
All microorganisms shown in Table 1 were obtained from the NBRC culture
collection (NITE Biological Resource Center, Kisarazu, Japan). Cultivation was
performed using ISP2 liquid medium at 371C for 24h. The disc diffusion assay
using a paper disc (i.d. 6 mm, thin type for 15mg; and i.d. 8 mm, thick type for
0.92(d, 6.9)
0.77 (d, 6.9)
’
7.52 (d, 15.1)
6.80 (d, 15.1)
5
0mg) was performed to determine the antimicrobial activity of 1 and 2.
’
’
ACKNOWLEDGEMENTS
’
This study was supported by research funds from the Takeda Science
Foundation, the Astellas Foundation for Research on Metabolic Disorders, the
Foundation of Hattori Hokokai and The Kurata Memorial Hitachi Science and
Technology Foundation. We appreciate Mr Hidemi Nagata for providing the
soil samples.
’
ꢀ
’
36.3
26.4
ꢀ
2.76 (br)
2.44 (br)
’
’
which was subjected to an open column chromatography (Mitsubishi
Chemical, Tokyo, Japan, CHP20P, 3ꢁ 5 cm), eluting with 50ml each of 20%
MeOH, 60% MeOH and MeOH. The MeOH fraction was subjected to reverse-
phase HPLC using an ODS column (Nacalai Tesque, Kyoto, Japan, Cosmosil
C18-MSII 4.6ꢁ 250 mm) with an isocratic elution of 85% MeCN containing
1
2
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makinolide (1, 3.3mg) and JBIRꢀ100 (2, 2.0mg).
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