Stalobacin: Discovery of Novel Lipopeptide Antibiotics with Potent Antibacterial Activity against Multidrug-Resistant Bacteria

Article abstract of DOI:10.1021/acs.jmedchem.0c00295

A novel lipopeptide antibiotic, stalobacin I (1), was discovered from a culture broth of an unidentified Gram-negative bacterium. Stalobacin I (1) had a unique chemical architecture composed of an upper and a lower half peptide sequence, which were linked via a hemiaminal methylene moiety. The sequence of 1 contained an unusual amino acid, carnosadine, 3,4-dihydroxyariginine, 3-hydroxyisoleucine, and 3-hydroxyaspartic acid, and a novel cyclopropyl fatty acid. The antibacterial activity of 1 against a broad range of drug-resistant Gram-positive bacteria was much stronger than those of last resort antibiotics such as vancomycin, linezolid, and telavancin (MIC 0.004-0.016 μg/mL). Furthermore, compound 1 induced a characteristic morphological change in Gram-positive and Gram-negative strains by inflating the bacterial cell body. The absolute configuration of a cyclopropyl amino acid, carnosadine, was determined by the synthetic study of its stereoisomers, which was an essential component for the strong activity of 1.

Full text of DOI:10.1021/acs.jmedchem.0c00295

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Article
Stalobacin: Discovery of Novel Lipopeptide Antibiotics with
Potent Antibacterial Activity against Multidrug-resistant Bacteria
Kouhei Matsui, Yukiko Kan, Junko Kikuchi, Keisuke Matsushima, Miki
Takemura, Hideki Maki, Iori Kozono, Taichi Ueda, and Kazuyuki Minagawa
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00295 • Publication Date (Web): 07 May 2020
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Stalobacin: Discovery of Novel Lipopeptide
Antibiotics with Potent Antibacterial Activity
against Multidrug-resistant Bacteria
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Kouhei Matsui,*,†,‡ Yukiko Kan,‡ Junko Kikuchi,‡ Keisuke Matsushima,† Miki Takemura,‡ Hideki
Maki,‡ Iori Kozono,‡ Taichi Ueda,‡ and Kazuyuki Minagawa†
†Pharmaceutical Research Center, Shionogi TechnoAdvance Research Co., Ltd., 3-1-1, Futaba-
cho, Toyonaka-shi, Osaka 561-0825, Japan
‡Pharmaceutical Research Center, Shionogi & Co., Ltd., 3-1-1, Futaba-cho, Toyonaka-shi, Osaka
561-0825, Japan
KEYWORDS: lipopeptide, antibiotics, natural product, multidrug-resistant bacteria,
carnosadine
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ABSTRACT
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A novel lipopeptide antibiotic, stalobacin I (1) was discovered from a culture broth of an
unidentified Gram-negative bacterium. Stalobacin I (1) had a unique chemical architecture
composed of an upper and a lower half peptide sequence, which were linked via a hemiaminal
methylene moiety. The sequence of 1 contained an unusual amino acid, carnosadine, 3, 4-
dihydroxyariginine, 3-hydroxyisoleucine and 3-hydroxyaspartic acid, and a novel cyclopropyl
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fatty acid. The antibacterial activity of 1 against a broad range of drug-resistant Gram-positive
bacteria was much stronger than those of “last resort” antibiotics such as vancomycin, linezolid,
and telavancin (MIC 0.004-0.016 g/ml). Furthermore, compound 1 induced a characteristic
morphological change in Gram-positive and Gram-negative strains by inflating the bacterial cell
body. The absolute configuration of a cyclopropyl amino acid, carnosadine was determined by
synthetic study of its stereoisomers, which was an essential component for the strong activity of
1.
INTRODUCTION
Bacterial infection therapy has become difficult in recent years due to the emergence of
antimicrobial resistance, leaving fewer options for medical care.1,2 Most antibiotics on the market
have a specific target in bacteria and interfere with only one particular cellular function, such as
cell wall synthesis, protein or RNA synthesis, DNA replication or energy metabolism.
Unfortunately, bacteria can become intrinsically resistant to these antibiotics by changing the
affinity of the target molecule by genetic mutation, and multidrug-resistant (MDR) bacteria lose
their susceptibility to multiple principal antibiotics. The emergence of antibiotic resistance and the
spread of antibiotic-resistant pathogens form a dynamic process and cause serious health
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problems.3,4 Therefore, continuous investment in research and development of new generation
antibiotics is even now necessary to overcome the menace of multidrug-resistant bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant bacteria.5-7
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Despite continuous efforts to fight antibiotic resistance, only a few antibiotics with new modes of
action against bacteria have become available or have entered late-stage clinical trials over the past
decade.8,9 Historically, natural resources have played significant roles in the field of biomedical
applications by providing diverse bioactive compounds. Among them, a group of lipopeptides has
offered many important antibiotics with new action mechanism, which have advantages compared
to peptides of improved access to the target on cell membrane and cell wall, and metabolic
stability.10 For example, daptomycin, a cyclic lipodepsipeptide with a decanoyl side-chain, was
produced from the Gram-positive bacterium Streptomyces roseosporus. It exhibits potent
antibacterial activity by disrupting cell membrane functions of bacteria and is used as a last resort
agent to treat MRSA infections.11,12 Echinocandins are a class of cyclic lipopeptide compounds
derived from fungi, and have potent antifungal activity, with caspofungin being the most widely
marketed as an antifungal drug. The activity of echinocandins is dependent on the strong inhibition
of -glucan synthesis in the fungal cell wall.13,14 Polymyxins, which are produced by Gram-
positive bacteria such as Paenibacillus polymyxa, are efficient for a wide range of Gram-negative
bacteria, though their serious side effects cannot be ignored. The mechanism of action of
polymyxins has been deduced to be membrane disruption due to interaction between the cationic
polymyxin and the anionic bacterial outer membrane, resulting in a detergent-like activity.15,16
In this article, we report the discovery of a novel lipopeptide antibiotic, stalobacin I (1), from a
culture broth of an unidentified Gram-negative bacterium. Stalobacin I (1) had a unique chemical
architecture composed of an upper and a lower half peptide sequence, which were linked via a
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hemiaminal methylene moiety. The antibacterial activity of 1 against a broad range of drug-
resistant Gram-positive bacteria was much stronger than those of “last resort” antibiotics such as
vancomycin, linezolid, and telavancin (MIC 0.004-0.016 g/ml).
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RESULTS AND DISCUSSION
Successive investigation of bioactive metabolites from collections of bacteria revealed one fraction
exhibiting broad and significant antibacterial activity against MRSA from a culture broth of an
unidentified Gram-negative bacterium, PBJ-5360, which was isolated from soil collected in Kyoto,
Japan. Bioassay-guided purification by ODS HPLC led to isolation of nine active compounds,
stalobacin A-I. However, the productivity of stalobacins in 3 L culture broth was on the g order.
Improvement of the fermentation condition by changing the culture medium composition, growth
temperature and aeration made it possible to obtain a sufficient amount of stalobacin I (1), which
was the most abundant analog, for structure determination and pharmacological experiments. In
the scale-up experiment, PBJ-5360 strain was cultured on the 125 L scale using a jar fermenter,
1
and LC-MS and H NMR profiling data guided the production and purification of 1. First,
compound 1 in culture broth was adsorbed onto resin, and the extract from the resin was purified
by ODS and ODP column to finally afford 38 mg of 1 as a white amorphous substance.
Compound 1 possessed a molecular formula of C57H92N14O22 deduced from the protonated
molecule [M + H]+ at m/z 1325.6595 by HR-(+)-LSIMS, which was also supported by elemental
1
13
analysis (supporting information S5). Based on H- and C NMR analysis in DMSO-d6 at 40
degrees, the peptidic nature of 1 was evident from a large number of signals for exchangeable
amide NH protons (δH 7.47−8.53) and for α-amino acid protons in the deshielded region (δH
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3.76−5.61) along with a large number of carbonyl carbons (δC 170.3−174.3) attributable to
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ester/amide functionalities. Furthermore, the large H signal at around δH 1.26 revealed the
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presence of a long aliphatic chain.
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Compound 1 hydrolyzed with HCl was analyzed by standard amino acid analysis and Marfey’s
method, 17,18 and three proteinogenic amino acids, L-Asp, L-Gly and L-Ser, two nonproteinogenic
amino acids, N-methyl-L-aspartic acid (N-MeAsp) and L-threo-3-hydroxyaspartic acid (HyAsp),
and two unknown amino acids, X1 and X2, were detected. The unknown amino acids X1 and X2
were purified by ion-exchange chromatography, and X1 was determined to be 3-
hydroxyisoleucine (HyIle) by MS and the NMR data analysis of 1 as described below. On the other
hand, the planar structure of the isolated X2 was elucidated to be identical to that of
carnosadine19,20 isolated from cyanobacteria by Shiba et al. based on HR-(+)-LSIMS and NMR
data analysis, but its NMR spectrum was not identical to that of (1S, 2S)-carnosadine, which
suggests that X2 may be a stereoisomer of carnosadine.
Next, we tackled the overall structural elucidation of 1 by 2D-NMR. Since the NMR analysis was
very difficult due to signal overlapping and broadening, we determined the structure of 1, step-by-
step: 1) elucidation of the partial structure of 1 by NMR analysis of 1 and 2) elucidation of the
overall structure of 1 by NMR analysis of derivative 2, which lacks a guanidyl moiety in the
carnosadine part.
Taking various solvent and salt forms of 1 into consideration, the NMR measurement of formate
of 1 in DMSO-d6 at 40 degrees gave a spectrum that could be evaluated for partial structures of 1
(Figure 1). The measurement at high temperature was effective for the improvement of signal
broadening. Extensive analysis of the 2D-NMR experiments of 1, including COSY, TOCSY,
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HSQC, HMBC and ROESY, revealed the presence of carnosadine lactam, whose -amino group
was substituted by a hemiaminal methylene group. Furthermore, carnosadine lactam was linked to
the C-3 of HyIle through a hemiaminal methylene group. On the other hand, interpretation of the
2D-NMR clarified the structure of a fatty acid part constituted of a 4-cyclopropyl-2-hydroxy-3-
butenoic acid moiety, an isolated olefin between alkyl chains and a terminal long alkyl chain. Both
geometries of a cyclopropane and an olefin in the 4-cyclopropyl-2-hydroxy-3-butenoic acid moiety
were determined as having the trans configuration based on ROE signals (from H-6 to H-8 and
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H-5, from H-6 to H-4, from H-4 to H-7) and the coupling constant at H-3 and H-4 (15.3 Hz),
respectively. The geometry of the isolated olefin was assigned a cis configuration on the basis of
the chemical shifts of the allylic carbons (δC 26.50 and 26.52).
Carnosadine
H
fatty acid
NH2
NH
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N
n
m
N
O
H
O
7
5
H
O
H
HyIle
6
1
H
N
OH
H
O
Figure 1. Partial structures of stalobacin I (1) with key HMBC (one-way arrows) and ROE (two-
way arrows) correlations.
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As for the other partial structure, NMR analysis of 1 did not give a clear solution due to
signal overlapping and broadening. Therefore, we tried structural conversion to facilitate
the NMR analysis. The reaction of 1 using EtNH2 gave compound 2 with a molecular
formula of C56H90N12O22 deduced from HR-(+)-LSIMS analysis, which was identical to a
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CN2H2 unit deletion variant of 1. Since the H and C NMR signals in the carnosadine
lactam part of 2 were shifted to the high magnetic field side compared to those of 1, and
the guanidyl carbon of the carnosadine lactam part was absent from 2, compound 2 was
concluded to be the CN2H2 unit deletion variant of the carnosadine lactam part of 1 as
shown in Figure 2. The NMR spectrum in DMSO-d6 of 2 at 50 degrees gave separated and
sharp signals compared to 1, and the analysis was considerably easier than that of 1.
Detailed analysis of the 2D-NMR experiments of 2, including COSY, TOCSY, HSQC,
HMBC and NOESY, revealed an upper and a lower half substructure, which were
connected through a hemiaminal carbon.
In the upper half substructure, the NOESY signal between HyAsp H-2/N-MeAsp N-CH3
and HMBC correlation from N-MeAsp N-CH3 to HyAsp CO(δC) revealed a HyAsp-N-
MeAsp sequence. The N-terminal amine of this sequence was amidated to 2-hydroxy-3-
butenoic acid moiety based on the NOESY signal between HyAsp H-2/butenoic acid H-2
and HMBC correlation from HyAsp H-2 to butenoic acid CO(δC). Furthermore, the amide
linkage between the C-terminal of this sequence and -amino group of carnosadine lactam
was elucidated by the HMBC correlation from the hemiaminal methylene proton. In the
lower half substructure, the presence of two nonproteinogenic amino acids was elucidated,
3,4-dihydroxyarginine (3,4-diHyArg) and HyIle by analysis of the 2D-NMR. Furthermore,
HMBC correlations from H-2 to carbonyl carbons of adjacent amino acids along with
NOESY correlations between H-2 and NH protons of adjacent amino acids allowed us to
establish the sequence as Asp-3,4-diHyArg-HyIle-Gly-Ser. Finally, the planar structure of
1 was elucidated as shown in Figure 2 by the NMR analysis of 1 and 2.
The location of an isolated olefin between alkyl chains in the fatty acid part was elucidated
by a chemical degradation experiment of 1. Oxidative cleavage by the OsO4-NaIO4 system
is a highly specific reaction for cleavage of olefin under a mild condition.21,22 An isolated
olefin of 1 was oxidized by microencapsulated osmium tetraoxide MC OsO4 to afford a
major compound, which was deduced to be a dihydroxyl derivative of 1. NaIO4 oxidation
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of a dihydroxyl derivative followed by NaBH4 treatment also gave an alcohol 3 (Figure 3).
The other olefin adjacent to the cyclopropane was speculated to not be oxidised at the first
oxidation step due to steric hindrance. These findings elucidated the presence of five
methylene units from C8-C12 and five methylene units from C15 to C19, which were
connected through an isolated olefin.
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Next, we tried to clarify the absolute configuration of carnosadine lactam in 1. (1S, 2S)-
Carnosadine was first isolated from a culture broth of the cyanobacteria Grateloupia
carnosa by Shiba et al.19,20 Asymmetric synthesis of (1S, 2S)-carnosadine and its
stereoisomers were reported by Husson et al. and Burgess et al.23,24 The planar structure of
the amino acid X2 obtained from the hydrolysate of 1 matched that of carnosadine, but its
NMR spectrum was not identical to that of (1S, 2S)-carnosadine, which suggested X2 to be
a stereoisomer of (1S, 2S)-carnosadine. Therefore, we synthesized (1S, 2R) and (1R, 2S)-
carnosadine to identify the absolute stereochemistry of X2. The corresponding chiral
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intermediates prepared according to a previous report were converted to (1S, 2R) and
(1R, 2S)-carnosadine in three steps as shown in Supporting Information S27. Consequently,
the NMR spectrum, specific rotation, CD spectrum and retention time on HPLC analysis
of X2 were in good agreement with those of the synthetic (1S, 2R)-carnosadine (Figure 4).
Finally, the overall structure of 1 was determined as shown in Figure 2, although the
absolute stereochemistries of eight asymmetric carbons were not clear: C-2, C-3 and C-4
in the 3,4-diHyArg residue, C-2 and C-3 in HyIle, and C-2 and trans-cyclopropane carbons
in the fatty acid part.
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fatty acid
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7
8
NH2
NH
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1
: R=
Carnosadine
HyAsp
2
: R=H
OH
H
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H
COOH
H
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7
CH3
*
O
H
O
H
N
N
R
6
5
1
*
N
N
H
H
H
H
O
O
OH
HO
-MeAsp
HyIle
O
N
O
HOOC
OH
Ser
O
O
Asp
H
N
H
N
H2N
N
N
COOH
H
H
H
H
O
O
Gly
HO
OH
3,4-diHyArg
NH
HN
NH2
Figure 2. Structure of stalobacin I (1) and its derivative 2, and key HMBC (one-way arrows) and
NOE (two-way arrows) correlations in 2. The absolute configuration of the carbons with asterisk
was not clarified, though the relative configuration of them was clear.
1) MC OsO4
OH
2) NaIO4
3) NaBH4
3
H
1
*
H
O
H
*
H
OH
Figure 3. Oxidative degradation of stalobacin I (1)
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H
H2N
HO
N NH2
1 2
NH
O
Figure 4. Structure of (1S, 2R)-carnosadine
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The antibacterial activity of 1 was determined based on the minimum inhibitory
concentration (MIC) for 15 Gram-positive and 2 Gram-negative bacteria including drug-
resistant strains. The MICs of 1 for all Gram-positive bacteria reached 0.004-0.016 g/ml,
which were much lower than that of “last resort” antibiotics against serious and multidrug-
resistant infections caused by Gram-positive bacteria, vancomycin, linezolid, and
telavancin (Table 1).25-30 It was notable that 1 showed 125-250-fold stronger antibacterial
activity against S.aureus SR3637(H-MRSA) than these antibiotics. Furthermore,
compound 1 also had a potent antimicrobial effect on vancomycin-resistant strains, which
have become new challenges in antibacterial treatment. On the other hand, the activity
against Gram-negative bacteria such as E. coli and K. pneumoniae tended to be weaker than
that against Gram-positive bacteria. This phenomenon may result from the poor
permeability of 1 through the outer membrane of Gram-negative bacteria and does not
contradict the fact that 1 is produced by a Gram-negative bacterium. Derivative 2 lacking
a guanidyl moiety in the carnosadine part showed much weaker antibacterial activity than
1 against every Gram-positive bacterium. In particular, the activity against H-MRSA of 2
was 125-fold weaker than that of 1. These findings indicate that the carnosadine part of 1
constitutes the pharmacophore for its strong antibacterial activity. Compound 1 exhibited
low cytotoxicity against murine leukemia P388 cells and L1210 cells (IC50 > 12.5 g/ml,
data not shown).
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Table 1. Antibacterial activity of stalobacin I (1) and its derivative 2
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MIC (g/ml)
8
9
1
2
0.5
0.5
1
Vancomycin
Linezolid
Telavancin
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S.aureus SMITH
0.004
0.004
0.008
0.004
0.004
0.008
0.016
0.004
0.004
0.004
0.004
0.004
≦0.004
≦0.004
≦0.004
4
1
2
2
0.5
S.aureus RN4220
1
0.5
S.aureus SR3637 (H-MRSA) 33
S.aureus HIP11714 (vanA)
S.aureus HIP11983 (vanA)
E.faecalis SR1004
2
2
1
0.5
1
>64
>64
1
2
4
2
4
>64
>64
>64
4
4
1
E.faecalis SR7914 (vanA) 33
E.faecalis SR23630 (vanB)
E.faecium SR16972
>64
32
2
4
1
2
2
2
0.5
E.faecium SR7940 (vanA)
E.faecium SR23598 (vanB)
E.faecium SRM1101 (c_vanB)
S.pneumoniae Type I 34
S.pneumoniae SR11031 (PRSP) 35
S.pneumoniae tupelo (VTSP) 36
E.coli NIHJ JC-2
2
>64
>64
>64
0.5
0.25
0.25
>64
>64
2
8
1
2
0.5
1
2
8
0.25
0.25
1
≦0.063
≦0.063
≦0.063
ND
0.5
1
≦0.004
4
2
ND
ND
K.pneumoniae SR135
1
ND
MICs were determined by a microdilution broth method as recommended by the NCCLS with
cation-adjusted Mueller−Hinton broth (CA-MHB). Serial 2-fold dilutions of each compound were
prepared in appropriate broth, and the plates were inoculated with 5×104 CFU of each strain in a
volume of 0.1 mL. Plates were incubated at 35°C for 20 h, and then MICs were scored.
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Interestingly, compound 1 induced a characteristic morphological change in Gram-positive
and Gram-negative bacteria. As shown in Figure 5, compound 1 caused inflation of the
bacterial cell body of Gram-positive bacteria, S. aureus RN4220 and Gram-negative
bacteria E. coli JC-2. Cell wall synthesis inhibitors such as -lactam antibiotics also are
known to induce morphological change of S. aureus and E. coli,31,32 but the morphology
they induce seems to be qualitatively different from that induced by 1 (Supporting
Information S29). This action of 1 may have a correlation with the mechanism of its
antibacterial activity. The similar action of 1 against both Gram-positive and Gram-
negative bacteria suggests that both classes of bacteria possess a similar essential target
molecule that is attacked by 1. Furthermore, we found that it was difficult to acquire
stalobacin-resistant bacteria, which implies that the approach to stalobacin-target molecule
is valid for the creation of the new drug with high resistance barrier.
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Figure 5. Morphological change of S.aureus RN4220 and E.coli JC-2 induced by stalobacin I (1)
. All photomicrographs were taken at the same magnifications.
CONCLUSIONS
Bacterial pathogens inevitably develop severe resistance to every new antibacterial drug launched.
Here, a novel lipopeptide antibiotic, stalobacin I (1), was found to possess excellent antibacterial
activities against a broad range of bacterial species including methicillin-resistant Staphylococcus
aureus (MRSA) and vancomycin-resistant bacteria. To our knowledge, the activity of 1 against
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multidrug-resistant bacteria ranks in the strongest class among those of previous reported
antibiotics. Furthermore, compound 1 induced a morphological change in Gram-positive and
Gram-negative strains, causing inflation of the bacterial cell body. The unique chemical
architecture of 1 including unusual amino acids and fatty acid suggests that 1 has a specific target
essential for bacteria; natural products with a complex structure often interact with a specific target
in vivo. The mechanism of action of 1 is under investigation for the development of new
therapeutics against the menace of multidrug-resistant bacteria.
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EXPERIMENTAL SECTION
General information. Analytical HPLC experiments were conducted on an Agilent Infinity 1290
system, LC-MS experiments on an Agilent 6130 LC-MS system, preparative HPLC experiment
on a Shimadzu LC-20AD. HR-LSIMS were measured on a Hitachi M-90 mass spectrometer. NMR
spectra were recorded on a Varian INOVA 600 MHz spectrometer. Chemical shifts are given in
ppm (δ) relative to tetramethylsilane as an internal standard. IR spectra were recorded on a JASCO
DS-403G spectrophotometer. Optical rotations were measured on a JASCO (DIP-1000) digital
polarimeter. The purities of all tested compounds were confirmed to be greater than 95% by HPLC
analysis.
Isolation and taxonomy of the strain PBJ-5360. The strain PBJ-5360 was isolated from soil
collected in Kyoto, Japan. This bacterium could not be identified, despite comprehensive
examinations, including morphology, culture, physiological and biochemical properties, and 16S
rRNA analysis (Supporting Information S3).
Isolation of stalobacin I (1). An 800 ml medium (adjusted to pH 7) consisting of 1.0% glucose,
0.5% yeast extract (Difco) and tap water in a 2 L Erlenmeyer flask was inoculated with a seed
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strain of PBJ-5360 (kept at -80 degrees in a 2 ml vial), and the resultant mixture was subjected to
shaking cultivation at 180 rpm with 70 mm of shaking breadth at 28 degrees for 22 hours. The
culture (80 ml) was inoculated into 20 L of a medium (adjusted to pH 7) containing 1.0% glucose,
0.4% yeast extract (Difco), 1% malt extract (Difco), 0.1% polypeptone (Nihon Pharma) and tap
water in a 30 L jar fermenter, and the resultant mixture was cultivated with 14 L/min of aeration
by agitation at 200 rpm at 28 degrees for 21 hours. Next, 8 L of this culture was inoculated into
125 L of a medium (adjusted to pH 7) consisting of 2.0% soluble starch, 2.0% powder yeast, 1.5%
ß-cyclodextrin, 0.5% olive oil, 0.0008% Adecanol LG109 (Asahi Denka Kogyo) and tap water in
a 250 L jar fermenter, and the resultant mixture was cultivated with 65 L/min of aeration by
agitation at 350 rpm at 28°C for 72 hours.
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The culture broth obtained in the foregoing step was sterilized by adding 1.4 L of chloroform.
Next, 15 L of Amberlite XAD-7 (Organo) was added, and the resulting mixture was stirred for 3
hours for batch adsorption of the active substances onto the resin. After the resin was recovered
using a polyethylene mesh sheet, it was washed with water, put in a Büchner funnel and washed
with water, 30% methanol, and 50% methanol/20 mM phosphate buffer (pH 7.5). Next, the active
substances were eluted with 60% methanol/20 mM phosphate buffer (pH 7.5) and the fractions
showing antibacterial activity to S. aureus JC-1 were collected and concentrated in vacuo to 3 L.
The concentrate was washed with EtOAc, and the active substances were adsorbed on the
Amberlite XAD-7 column. The column was washed with water, and the active substances were
eluted with 30% methanol and 50% methanol. The fraction containing 1 was collected and
lyophilized to afford 3.89 g of crude product.
The crude product 1.82 g obtained in the foregoing step was dissolved in 60 ml of 20 mM
phosphate buffer (pH 7.5), and the solution was subjected to preparative high-performance liquid
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chromatography (HPLC) using an ODS column (column: YMC ODS S-15, 5.0 x 50 cm, eluent:
MeCN/20 mM phosphate buffer (pH 7.5) containing 50 mM Na2SO4 = 40/60, flow rate: 50 ml/min,
UV detection: 220 nm) to obtain a fraction containing 1 as the main ingredient. After removing
MeCN in vacuo, the concentrate was adsorbed on the Diaion HP-20 (Mitsubishi Chemical)
column, and the column was washed with water and eluted with 60% acetone aq. The eluent was
lyophilized to afford 126 mg of powder. Finally, the solution of the powder in 50 mM phosphate
buffer (pH 7.0) was subjected to preparative ODP HPLC (column: Asahipak ODP-90, 2.15 x 30
cm, eluent: 40% MeCN aq. containing 20 mM AcOH, flow rate: 8 ml/min, UV detection: 220 nm)
to obtain a pure fraction of 1. After the fraction was neutralized and concentrated, NaCl (final
concentration, 5%) was added, adjusting the solution to pH 7.5 with 1 N NaOH aq. The concentrate
was adsorbed on MCI GEL CHP20P (Mitsubishi Chemical), and the column was washed with
water and eluted with 70% MeOH aq. The eluent was concentrated and lyophilized to finally afford
38 mg of 1 as a white solid.
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The same procedure was repeated to finally obtain 216 mg of 1.
Synthesis of derivative 2. To a solution of 1 (20 mg, 0.015 mmol) in DMF (4 mL) was added
EtNH2 (0.2 ml, 70% in water), and the mixture was stirred at rt for 4 days. After removing the
solvent in vacuo, the residue was subjected to preparative ODS HPLC (column: Symmetry C18,
19 x 150 mm, eluent: 40% MeCN aq. containing 0.1% HCOOH, flow rate: 20 ml/min, UV
detection: 220 nm), and the pure fraction was lyophilized to obtain 8 mg of 2 as a white powder:
HRMS (ES) found m/z 1283.6378 [(M+H)+; calcd for C56H91N12O22 : 1283.6371].
Antibacterial activity evaluation. S. aureus HIP11714 and S. aureus HIP11983 were provided
by the network on antimicrobial resistance in S. aureus (http://www.narsa.net/content/default.jsp).
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Vancomycin-resistant Enterococcus faecalis SR7914 (VanA) and methicillin-resistant
Staphylococcus aureus SR3637 (H-MRSA) were clinical isolates collected from hospitals in
Japan.33
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ASSOCIATED CONTENT
Supporting Information.
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Identification of the strain PBJ-5360, characterization of stalobacin I (1), amino acid analysis of
stalobacin I (1) hydrolysate, characterization of derivative 2, oxidative degradation of 1, isolation,
synthesis and characterization of carnosadine, and observation of bacterial morphological change
by stalobacin I (1)
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-6-6331-7185. E-mail: kouhei.matsui@shionogi.co.jp
Notes
The first isolation of stalobacin I (1) was described in a patent, WO/1995/023812 A1. The detailed
structural analysis and the reassessment of antibacterial activity against various drug-resistant
bacteria of 1 were evaluated in this article for the first time. The authors declare no competing
financial interest.
ACKNOWLEDGEMENTS
We acknowledge Tatsuo Tanimoto and his co-workers (Shionogi & Co., Ltd.) for their help with
the purification of the stalobacins. We thank Kouichi Uotani, Miyako Okane and Merime Oota
(Shionogi & Co., Ltd.) for evaluating the antibacterial activities of the synthesized analogues.
ABBREVIATIONS
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HR-LSIMS, high-resolution liquid secondary ion mass spectrometry; S. aureus, Staphylococcus
aureus; E. faecalis, Enterococcus faecalis; E. faecium, Enterococcus faecium; S. pneumoniae,
Streptococcus pneumoniae; E. coli, Escherichia coli; K. pneumoniae, Klebsiella pneumoniae;
MIC, minimum inhibitory concentration; CFU, colony forming unit; ODS, octadecyl silyl; ODP,
octadecyl polymer
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