Kribellosides, novel RNA 5′-triphosphatase inhibitors from the rare actinomycete Kribbella sp. MI481-42F6

Article abstract of DOI:10.1038/ja.2016.161

Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5′-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5′-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5′-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5′-triphosphatase) from Saccharomyces cerevisiae in vitro with IC₅₀s of 5-8 μM and show antifungal activity with MICs ranging from 3.12 to 100 μg ml^-1 against S. cerevisiae.

Full text of DOI:10.1038/ja.2016.161

The Journal of Antibiotics (2017), 1–8
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
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ORIGINAL ARTICLE
Kribellosides, novel RNA 5′-triphosphatase inhibitors
from the rare actinomycete Kribbella sp. MI481-42F6
Masayuki Igarashi1, Ryuichi Sawa1, Manabu Yamasaki1, Chigusa Hayashi1, Maya Umekita1, Masaki Hatano1,
Toshinobu Fujiwara1,3, Kiyohisa Mizumoto2 and Akio Nomoto1
Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping
enzyme repressors are therefore candidate antifungal drugs. The 5ʹ-guanine-N7 cap structure of mRNAs are an essential feature
of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA.
Inhibitors of the RNA 5ʹ-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as
Candida. We discovered a new RNA 5ʹ-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites
from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p
(RNA 5ʹ-triphosphatase) from Saccharomyces cerevisiae in vitro with IC50s of 5–8 μM and show antifungal activity with MICs
ranging from 3.12 to 100 μg mlÀ1 against S. cerevisiae.
The Journal of Antibiotics advance online publication, 11 January 2017; doi:10.1038/ja.2016.161
INTRODUCTION
homodimerization of a domain.5 In yeast such as S. cerevisiae and
Candida albicans, the triphosphatase and guanylyltransferase are
encoded by distinct genes whose protein products form a non-
covalent complex. By contrast, in mammals and plants, the tripho-
Candidiasis is a fungal infection caused by yeasts of the genus Candida.
Candida is the fourth most common cause of healthcare-associated
bloodstream infections. These infections predominantly affect immu-
nocompromised patients, including those with AIDS or cancer, or
those who have received organ transplants. Currently, there are only a
few systemic antifungal drugs (amphotericin B, flucytosine, several
azole drugs and echinocandins) that are available for the treatment of
such invasive candidiasis, and not all of these are satisfactory in terms
of efficacy, toxicity, their antifungal spectra, or the possibility of drug
resistance. There is thus an urgent need for the discovery and
development of novel anti-Candida drugs, especially those which have
a different mode of action from the currently available drugs.
The 5′ guanine-N7 cap has been found to be an essential feature
of all eukaryotic organisms examined to date and is the first
co-transcriptional modification of cellular pre-messenger RNA.1–3
The first step of cap formation is the removal of γ-phosphate from
the RNA 5ʹ-triphosphate end of newly synthesized RNA to generate a
diphosphate end by RNA 5ʹ-triphosphatase. Then the GMP moiety of
GTP is transferred to the 5ʹ-diphosphate terminus by mRNA
guanylyltransferase. After these two consecutive reactions, methylation
at the guanine-N7 position catalyzed by mRNA (guanine-N7-)
methyltransferase follows. Saccharomyces cerevisiae RNA 5ʹ-tripho-
sphatease (Cet1p) is a member of the divalent cation-dependent
triphosphatase family observed in protozoa, eukaryotic viruses and
sphatase and guanylyltransferase occur in
chain. Although the guanylyltransferase domain is conserved across
evolution, the triphosphatase domain is metal-independent
a single-polypeptide
a
enzyme that shares structural homology with the cysteine phosphatase
superfamily.8–10
Thus, the capping enzymes of yeast differ greatly from the capping
enzymes of mammals, both structurally and mechanistically, and
inhibitors directed against Cet1p may have a fungicidal effect and may
be active against clinically important pathogenic yeasts.
During the course of our screening program for new Cet1p
inhibitors from metabolites of actinomycetes using a Cet1p enzyme
assay system, we found that a strain of Kribbella sp. MI481-42F6
produced new antibiotics, namely kribellosides (Figure 1a,b).
Kribellosides showed moderate RNA 5ʹ-triphosphatase inhibitory
activities with moderate anti-yeast activities against S. cerevisiae.
In this paper, we describe the isolation, structural determination
and biological activity of new RNA 5ʹ-triphosphatase inhibitors,
kribellosides.
RESULTS AND DISCUSSION
Taxonomy of the antibiotic-producing strain
fungi.4–7 The X-ray structure of Cet1p revealed the location of two Strain MI481-42F6 was isolated from a soil sample collected at
independent active sites within parallel tunnels that are formed by Nerima-ku, Tokyo, Japan. This strain formed well-branched substrate
1Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan and 2Department of Biochemistry, School of Pharmacy, Kitasato University, Tokyo, Japan
3Current address: Department of Biochemistry, Faculty of Pharmacy, Kindai University, Osaka, Japan.
Correspondence: Dr M Igarashi, Institute of Microbial Chemistry (BIKAKEN), Laboratory of Microbiology, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan.
E-mail: igarashim@bikaken.or.jp
This paper is dedicated to Professor Dr Satoshi Ōmura for his Nobel Prize in Physiology or Medicine 2015.
Received 14 October 2016; revised 16 November 2016; accepted 24 November 2016

Novel Cet1p inhibitors from actinomycete
M Igarashi et al
2
O
O
OH
O
O
OH
O
O
O
O
OH
OH
HO
O
O
HO
O
O
OH
OH
OH
OH
OH
OH
HO
HO
Kribelloside A (1)
Kribelloside C (3)
OH
OH
O
O
OH
O
O
OH
O
O
O
O
OH
OH
OH
OH
HO
HO
Kribelloside B (2)
Kribelloside D (4)
OH
OH
Figure 1 (a) Structure of kribellosides A, B, C and D. (b) Electron micrograph of the kribelloside producer Kribbella sp. MI481-42F6. Kribbella sp.
MI481-42F6 was grown on oatmeal agar (ISP medium No. 3) for 14 days at 30 °C. Bar, 1 μm.
mycelia and straight to flexuous aerial mycelia. Mature spore chains Physico-chemical properties and structure determination of the
consisted of 8 to 10 or more spores. Each spore was oval in shape with kribellosides
a smooth surface and was 0.6 × 0.8–0.9 × 1.1 μm in size. The substrate The physico-chemical properties of compounds 1, 2, 3 and 4 are
mycelia were pale yellow to pale yellowish brown, whereas aerial shown in Supplementary Table S1. The optical rotations of
mycelia were white. Diaminopimelic acid isomers were of the LL-type. compounds 1–4 were +103.8°, +61.7°, +95.7° and +50.4°, respectively
The partial 16 S ribosomal RNA gene sequence (1450 bp) was (c 0.5, MeOH). Their UV spectra showed no characteristic absorption.
determined and submitted to the GenBank/EMBL/DDBJ database The molecular formulas of compounds 1–4 were determined to be
under accession number LC049957. The gene sequence showed high C30H56O14, C24H46O9, C31H58O14 and C25H48O9, respectively, by
similarity with those of the genus Kribbella, such as Kribbella high-resolution electrospray ionization mass spectrometry (HRESI-
aluminosa (JCM 14599T, 1419/1432 bp, T: Type strain, 99.1%)11 and MS, negative-ion mode), revealing a difference of CH2 between 1, 2
K. solani (JCM 12205T, 1433/1453 bp, 98.6%).12
and 3,
4
and C6H10O5 between 1,
3
and 2, 4. The
These phenotypic and genotypic data suggested that strain LC-HRESI-MS indicated that 1 and 2 share the same in source
MI481-42F6 belongs to the genus Kribbella. Therefore, the strain fragment of m/z 303.2896 (C18H39O3) and 3 and 4 possessed the
was tentatively designated as Kribbella sp. MI481-42F6. Detailed fragment of m/z 317.3052 (C19H41O3), as shown in Figure 2a.
taxonomic studies of strain MI481-42F6 are now underway.
The NMR data for the kribellosides are summarized in Table 1.
Compound 1 had 1 carbonyl, 2 anomers, 9 oxymethines, 1 sp3
methine, 4r oxygenated methylenes, 11 methylenes and 2 methyl
Fermentation and isolation of kribellosides
Yeast Cet1p requires magnesium ions for RNA 5′-triphosphatase carbons, as determined by 13C NMR and DEPT135 spectra. The 1H1H
activity, but has been reported to exert the nucleoside triphosphatase COSY and TOCSY spectra of compound 1 established the five parts
activity in the presence of manganese ions.13 Therefore, we monitored shown in Figure 2b. Correlations were observed from the oxymethine
kribellosides using an in vitro enzyme assay to determine their of δH 3.96 (H2) to the oxymethylene at δH 3.40, 3.83 (H1) and
inhibitory effect on Cet1p nucleoside triphosphatase activity during oxymethylene at δH 3.46, 3.49 (H3), suggesting a glyceryl moiety. The
the purification process. The fermentation of kribellosides was cross peaks observed from the anomer proton at δH 4.82 (H1ʹ) to
performed by culturing in a 500-ml baffled Erlenmeyer flask contain- oxymethine at δH 4.07 (H5ʹ) within three oxymethines (δH 3.51 (H2ʹ),
ing 110 ml of a producing medium with rotary shaking. The
δH 3.91 (H3ʹ) and δH 3.71 (H4ʹ)), and from another anomer proton at
fermentation broth (4 l) was separated into the mycelial cake and δH 5.27 (H1ʹʹ) to the oxymethylene at δH 3.65 and 3.81 (H6ʹʹ) within
the supernatant by centrifugation. The mycelial cake was extracted four oxmethines (δH 3.37 (H2ʹʹ), δH 3.62 (H3ʹʹ), δH 3.26 (H4ʹʹ) and δH
with MeOH (1 l). The extract, including an active substance, was 3.72 (H5ʹʹ)) suggested two sugar moieties. The remaining portion
sequentially extracted with BuOH and a mixture of CHCl3-MeOH- might be a branched long alkyl chain from the oxymethylene at
water (5:4:6) yielding a brown material (764 mg). The brown material δH 3.46 (H4) to the terminal branched dimethyl protons at δH 0.88
was subjected to chromatography on a low-pressure reversed-phase (H17, 18) revealed by the 13-methyl-1-tetradecanol moiety. The
(C18) column and was developed with MeOH:ammonium carbonate connectivities of these parts were established by HMBC spectroscopy,
(5 mM) at a ratio of 0:100, 60:40, 80:20 and 100:0. The active fractions the long-range couplings; H5ʹ/C1ʹ (anomer at δC 100.9) and carboxyl
were collected and further chromatography was performed by carbon at δC 175.2 (C6ʹ) and large proton-proton couplings from H2ʹ
reversed-phase HPLC and developed with acetonitrile/ammonium to H5ʹ (9.4–9.5 Hz) and ROEs between H3ʹ and H5ʹ revealed an
carbonate (5 mM). The active fractions were collected and concen- α-glucuronic acid moiety. Long-range couplings, H5ʹʹ/C1ʹʹ (anomer at
trated in vacuo to yield 79 mg of pure kribelloside A(1), 83 mg of δC 101.2) and large couplings from H2ʹʹ to H5ʹʹ (9.5 Hz) and ROEs
kribelloside B(2), 26 mg of kribelloside C(3) and 13 mg of kribelloside between H3ʹʹ and H5ʹʹ revealed an α-glucose moiety. The HMBC
D(4), respectively, as a colorless powder.
correlation of H1ʹʹ/C4ʹ established an α-1, 4 bond between glucose and
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Novel Cet1p inhibitors from actinomycete
M Igarashi et al
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Figure 2 (a) COSY, HMBC and NOE analyses of the kribellosides. (b) ESI/MS fragment analysis of the kribellosides.
glucuronic acid. Furthermore, long-range connectivities were observed above suggested a lack of glucose moiety in compound 1. Compound
from H1ʹ of the anomer proton of glucuronic acid to glyceryl 2 was found to possess 1 carbonyl, 1 anomer, 5 oxymethines, 1 sp3
oxymethylene of H1 and another glyceryl oxymethylene of H3 to methine, 3 oxygenated methylenes, 11 methylenes and 2 methyl
H4 of the 13-methyl-1-tetradecanol moiety. Taken together, these carbons from the 13C NMR and DEPT135 spectra. Furthermore,
results determined the planer structure of 1. The length of the 1D and 2D spectral analyses revealed three parts, namely the glyceryl,
branched alkyl long chain was also established by the ESI/MS in 13-methyl-1-tetradecanol and glucuronic acid, that were the same as
source fragment (m/z 303.2896 (C18H39O3)) as shown in Figure 2a.
in compound 1. The difference between compounds 1 and 2 was the
The structures of compounds 2–4 were determined in the same higher field shifts from H2ʹ to H5ʹ of glucuronic acid, as observed in
manner and compared with the data for compound 1. Comparisons of the 1H NMR spectrum. The same in source fragments of ESI/MS
the molecular formula and the 1H NMR spectra for 1 and 2 described spectra for compounds 1 and 2 also supported this structure
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Novel Cet1p inhibitors from actinomycete
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5
O
1) 3M HCl-MeOH/100oC/o.n.
OH
O
O
2) benzoyl chloride/CH2Cl2/0-40oC
O
OH
OH
O
HO
R
OH
O
O
O
3M HCl-MeOH/100oC/o.n.
OH
O
5
O
O
O
O
HO
6
O
OH
O
O
HO
HO
O
OH
OH
O
OH
1M HCl aq./85oC/o.n.
HO
O
O
OH
OH
OH
OH
HO
OH
Figure 3 Degradation study of kribelloside A (1).
(Figure 2a). Thus, the structure of 2 was determined as shown in
Figure 2. The structures of compounds 3 and 4 were similar to those
of 1 and 2 with the exception of one methylene. The 2D analyses of
compounds 3 and 4 revealed that they possessed 14-methyl-
1-pentadecanol instead of the 13-methyl-1-tetradecanol moiety of
compounds 1 and 2. This finding was supported by MS/MS analysis.
The structures of 3 and 4 were determined as shown in Figure 2b.
Table 2 Inhibitory activities of the kribellosides towards the RNA
5'-triphosphatase activities of Cet1p and hCap1
IC50 (μM)
Compound
Cet1p
hCap1
SIa
Kribelloside A
Kribelloside B
Kribelloside C
Kribelloside D
Suramin
6.8
6.3
3.6
4.5
0.7
26.1
17.4
16.1
9.3
3.8
2.8
4.4
2.1
7.9
Stereochemistry of the kribellosides
The absolute chemistry of the C2 of glyceryl ether and the two sugar
moieties of the kribellosides was determined by chemical degradations
and their derivatives, as shown in Figure 3. Methanolysis of compound
2 indicated an aglycon and sugar moiety. The reaction mixture was
partitioned by a solvent system of n-hexane-MeOH and the n-hexane
layer containing aglycon was derivatized to dibenzoate 5. Since the CD
spectrum of compound 5 indicated a negative cotton effect, the C2
was determined to be R by the dibenzoate chirality method.14 The
sugar moiety 6 described above was purified from the MeOH layer
and identified as methyl-β-D-glucofuranuronic acid γ-lactone by
comparing its NMR and CD spectra for the methanolysis product
of D-glucuronic acid. Finally, the absolute chemistry of the glucose
moiety in 1 was established to be D by the mutarotase-glucose oxidase
method.15 Taking all of these results into account, the absolute
chemistry of compounds 1 and 2 are shown in Figure 1. Compounds
3 and 4 showed the same stereochemistry, as indicated by the physico-
chemical properties and the NMR data.
5.4
Abbreviation: SI, selectivity index.
aThe SI as determined by the ratio of the IC50 values between hCap1 and Cet1p.
Biological activities of the kribellosides
Cet1p inhibitory activity. We examined the inhibitory activity of
kribellosides towards S. cerevisiae Cet1p using an in vitro enzyme assay.
Since, suramin has been reported to inhibit Cet1p activity, and under
our assay conditions it exhibited an IC50 value similar to that
previously reported (Table 2), confirming that our assay conditions
with [γ-32P]triphosphate-ended poly(A) were appropriate for evaluat-
ing the inhibitory effect of compounds on triphosphatase activity.
Upon treatment with kribelloside A, the hydrolysis of the γ phosphate
of triphosphate-ended RNA by Cet1p was inhibited in a dose-
dependent manner, with an IC50 of 6.8 μM (Figure 4 and Table 2);
whereas, kribellosides B, C and D inhibited Pi release from the RNA
with IC50 values of 6.3, 3.6 and 4.5 μM, respectively. Furthermore, we
evaluated the inhibitory potency of the compounds towards the RNA
triphosphatase activity of human mRNA capping enzyme, hCap.
Kribellosides A, B, C and D exhibited IC50 values of 26.1, 17.4, 16.1
and 9.3 μM, yielding selectivity indexes of 3.8, 2.8, 4.4 and 2.1,
respectively (Table 2). These results indicated that kribellosides are
novel inhibitors of S. cerevisiae RNA 5ʹ-triphosphatase. Glycosyl-
Kribellosides are novel compounds that belong to the ether lipids.
Ether lipids are known to be ubiquitous in the cell membranes of
mammals,16 marine organisms17 and archaea.18 These ether lipids,
however, are 1-O-alkyl lipids, whereas the 3-O-alkyl lipids in kribello-
sides are rare. Therefore, the 1-D-glucuronosyl-sn-glycerol 3-alkyl
ether is a unique structure among the ether lipids.
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Table 3 Antimicrobial activities of the kribellosides
MIC (μg ml− 1
)
Test organisms
A
B
C
D
S. cerevisiae W303a
S. cerevisiae W303α
S. cerevisiae SL101
S. cerevisiae HC101
S. cervisiae BY25929
100
100
50
50
50
100
100
100
50
12.5
25
50
12.5
12.5
3.12
50
25
25
12.5
12.5
reference. The mass spectra were recorded using an LTQ Orbitrap XL mass
spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). HPLC was
detected using an Evaporative Light Scattering Detector (Alltech Associates,
Inc., Deerfield, IL, USA).
Taxonomic studies of the producing strain MI481-42F6
Morphological properties were observed following incubation at 30 °C for
21 days on yeast extract-malt extract agar (ISP medium No. 2), oatmeal agar
(ISP medium No. 3), inorganic salts-starch agar (ISP medium No. 4), glycerol-
asparagine agar (ISP medium No. 5) and tyrosine agar (ISP medium No. 7).
Detailed observation of mycelial morphology was performed using a scanning
electron microscope S-570 (Hitachi High-Technologies) after strain MI481-
42F6 had been incubated on ISP medium No. 3 at 30 °C for 14 days. The type
of diaminopimelic acid isomers in the whole-cell hydrolysates was determined
by the method of Staneck and Roberts.22 Total DNA of strain MI481-42F6 was
prepared using a Genomic DNA Extraction Kit Mini (RBC Bioscience Co., New
Taipei, Taiwan) according to the manufacturer’s instructions. 16S rRNA
(positions 31–1524, Escherichia coli numbering system)23 was amplified by
PCR and sequenced. A search for the most closely related sequences was
performed using the BLAST algorithm in at the DNA Data Bank of Japan.
Fermentation of kribellosides
Figure 4 Inhibition of the RNA 5′-triphosphatase activity of Cet1p by
kribelloside A. Triphosphatase activity by Cet1(201-549)p. The reaction was
performed as described in the Materials and Methods using 32P-
triphosphate-ended poly(A). The reaction products were analyzed by PEI
cellulose TLC (upper panel) and quantified by phosphorimaging
(lower panel). The relative amount of the released [32P]Pi is shown as a
percentage of that of the untreated control (DMSO). Results are shown as
the means s.d. from three independent experiments. DMSO, dimethyl
sulfoxide; PEI, polyethyleneimine.
A slant culture of strain MI481-42F6 was inoculated into a 500-ml baffled
Erlenmeyer flask containing 110 ml of a seed medium consisting of 2% (w/v)
galactose, 2% (w/v) dextrin, 1% (w/v) Bacto Soytone (Becton Dickinson,
Franklin Lakes, NJ, USA), 0.5% (w/v) corn steep liquor (Kogo starch, Chiba,
Japan), 1% (w/v) glycerol, 0.2% (w/v) (NH4)2SO4 and 0.2% (w/v) CaCO3 in
deionized water (pH 7.4 before sterilization). The seed culture was incubated
on a rotary shaker (200 r.p.m.) at 30 °C for four days. The seed culture (2.5 ml)
of the strain was then transferred into a 500-ml baffled Erlenmeyer flask
containing 110 ml of a producing medium consisting of 2.0% glycerin, 2.0%
dextrin, 1.0% Bacto Soytone (Becton Dickinson), 0.3% yeast extract (Nihon
Pharmaceutical Co., Ltd, Tokyo, Japan), 0.2% (NH4)2SO4 and 0.2% CaCO3 in
deionized water (pH 7.4 before sterilization). The fermentation was carried out
on a rotary shaker (180 r.p.m.) at 27 °C for 7 days.
glycerol-alkyl ether has known biological activities, such as lipid
hapten activity,19 antimicrobial activity20 and the inhibition of cancer
metastasis.21 The 1-D-glucuronosyl-sn-glycerol 3-alkyl ether of kribel-
losides might be advantageous in its selectivity for the yeast enzyme
(Table 2).
Purification of kribellosides
The fermentation broth (4 l) was separated into the mycelial cake and
supernatant by centrifugation. The mycelial cake was extracted with MeOH
(1 l). The MeOH solution was removed in vacuo to yield a brown oil. The
brown oil, including an active substance, was extracted with BuOH and
concentrated in vacuo to give 1.4 g of a brown oil. The 1.4 g of brown oil was
extracted with a mixture of CHCl3-MeOH-water (5:4:6, 1 l). The upper layer
was collected and concentrated in vacuo to dryness yielding a brown material
(764 mg). The brown material was subjected to chromatography on a
low-pressure reversed-phase (20 × 250 mm, ODS-7515-12 A, Senshu Scientific
co., Tokyo, Japan) column developed with MeOH:ammonium carbonate
(5 mM) at a ratio of 0:100, 60:40, 80:20 and 100:0. The active fractions were
eluted with a ratio of 80:20 and 0:100, and concentrated in vacuo to give a pale
brown oil (427 mg). The active material was subjected to further chromato-
graphy on reversed-phase HPLC (Capcell Pak UG C18, 30 × 250 mm, Shiseido
Antimicrobial activity. We evaluated the antimicrobial activity of
kribellosides against S. cerevisiae. The anti-yeast activities of kribello-
sides are shown in Table 3. Kribellosides exhibited weak anti-
yeast activities against S. cerevisiae but no antibacterial activity against
Gram-positive and Gram-negative bacteria (data not shown).
MATERIALS AND METHODS
General experimental procedures
The optical rotations of the isolated kribellosides were measured using a P-1030
polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded using a U-2800
spectrophotometer (Hitachi High Technologies, Tokyo, Japan) and an
FT/IR-4100 Fourier transform infrared spectrometer (JASCO) was employed.
The 1H and 13C NMR spectra were measured using a JNM-ECA600 spectro-
meter (JEOL, Tokyo, Japan) at 25 °C using tetramethylsilane as an internal Co., Ltd., Japan) developed with acetonitrile:ammonium carbonate (5 mM) at
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Novel Cet1p inhibitors from actinomycete
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5:95 (0 min) to 50:50 (60 min) and 50:50 (80 min) at a flow rate of Fractions No. 26 and 27 were collected and concentrated to dryness to give
10 ml min− 1. The active fractions were collected and concentrated in vacuo
(6ʹ): 1H NMR (600 MHz, CD3OD) δ 4.92 (1H, s, H1), 4.91 (1H, dd, J = 4.6,
to yield 79 mg of pure kribelloside A (rt: 61–63 min), 83 mg of kribelloside B
16 mg of a colorless syrup of methyl-β-D-glucofuranosiduronic acid γ-lactone
(rt: 63–64 min), 26 mg of kribelloside C (rt: 65–66 min) and 13 mg of
(1H, s, H2), 3.33 (3H, s, OCH3). 13C NMR (CD3OD) δ 177.1 (C = O), 111.6
kribelloside D (rt: 67–68 min), respectively, as a colorless powder.
6.4 Hz, H4), 4.81 (1H, d, J =4.6 Hz, H3), 4.50 (1H, d, J = 6.4 Hz, H5), 4.18
(C-1), 84.7 (C-3), 79.4 (C-4), 78.5 (C-2), 70.5 (C-5), 55.7 (OCH3); HRESI/MS
[M+Na]+ m/z 213.0373 (calcd for C7H10O6Na, 213.0370).
Analytical procedure
The kribellosides in the fermentation broth and the various purification steps
were monitored by reversed-phase HPLC and silica gel TLC. HPLC was
Mutarotase-glucose oxidase method
performed on
a reversed-phase HPLC column (Capcell Pak UG C18,
Compound 1 (2.3 mg) was dissolved in 1 M HCl (1 ml) and the solution was
heated at 85 °C for 12 h. The reaction mixture was dried to remove the acid.
The residue was dissolved in water and washed with ethylacetate. The aqueous
solution was evaporated in vacuo. The residue was dissolved in water (200 μl,
1 × ), and its concentration was halved (2× ) for the sample solution. Standard
4.6× 150 mm, Shiseido, Japan; mobile phase, acetonitrile:ammonium carbo-
nate (5 mM) at a ratio of 45:55; flow rate, 1 ml min− 1; column temperature,
25 °C; detection, evaporative light scattering detector). Kribellosides A, B, C and
D were eluted at 6.5, 8.1, 10.3 and 12.8 min, respectively. A spot of each
antibiotic on TLC plates was detected by TLC (Kieselgel 60 F254, Art. No. 5715,
Merck) using a solution of molybdophosphoric acid and sulfuric acid in water.
The resulting Rf values for kribellosides A, B, C and D were 0.26, 0.36, 0.29 and
0.40, respectively, using the solvent system of CHCl3:MeOH:H2O at a ratio of
10:5:1.
D-glucose solutions (100, 500, 1000 and 2000 μg ml− 1
) and negative
controls of the L-glucose solution (1000 μg ml− 1) and D-glucuronic acid
(1000 μg ml− 1) were prepared. The detection of D-glucose was performed
using the mutarotase-glucose oxidase method.18 The sample, standard, negative
control and blank solutions (each 20 μl) were added to 3 ml of glucose CII kit
enzyme solution at 37 °C for 5 min. Then, the reaction mixtures were measured
at an absorption of 505 nm. For the standard D-glucose solutions (0, 500, 1000
and 2000 μg ml− 1), OD values of 0, 0.097, 0.217 and 0.462 were recorded, and
for the 1 × and 2 × sample solutions, OD values of 0.374 and 0.188 were
recorded, respectively. No absorption was detected for the 1000 μg ml− 1
solution of L-glucose and D-glucuronic acid.
(R)-3-((13-methyltetradecyl)oxy)propane-1,2-diyl dibenzoate (5)
Compound 2 (27.0 mg) was dissolved in 3 M of hydrogen chloride-methanol
and stirred at 100 °C for 14 h. The solution was added to 6 ml of n-hexane and
partitioned. The n-hexane layer was dried to yield 10.3 mg of crude aglycon.
The methanol layer was dried and re-dissolved in 100 ml of methanol and
1.8 ml of acetonitrile and then centrifuged at 2500 r.p.m. for 5 min. The
supernatant was concentrated and subjected to hydrophilic interaction liquid
chromatography (PC HILIC, 20× 250 mm, Shiseido, 97% acetonitrile). The
eluent of fraction No. 18 containing aglycon was combined with the residue of
the n-hexane layer, then evaporated in vacuo to yield 13.0 mg of crude aglycon.
The residue was dissolved in 1.5 ml of dichloromethane and benzoyl chloride
(14.3 mg, Tokyo Chemical Industry Co. Ltd.) and potassium carbonate
(9.0 mg) were added and stirred at 0 °C to 40 °C gently for 13 h. The reaction
mixture was filtered and subjected to preparative TLC (Kieselgel 60 F254, Art.
No. 5715) developed with n-hexane:ethylacetate at a ratio of 2:1 to give 1.5 mg
of compound 5 as a colorless syrup.
(R)-3-((13-methyltetradecyl)oxy)propane-1,2-diyl dibenzoate (5): colorless
syrup; 1H NMR (CDCl3, 600 MHz) δ 8.05 (2H, m, Ph), 8.02 (2H, m, Ph), 7.56
(1H, m, Ph), 7.55 (1H, m, Ph), 7.43 (2H, m, Ph), 7.42 (2H, m, Ph), 5.59
(1H, m, H2), 4.68 (1H, dd, J = 3.7, 11.9 Hz, H1a), 4.61 (1H, dd, J = 6.5,
11.9 Hz, H1b), 3.79 (1H, dd, J = 5.3, 10.5 Hz, H3a), 3.76 (1H, dd, J= 5.2,
10.5 Hz, H3b), 3.50 (1H, m, H1’), 1.24 (1H, m, H2’), 1.31 (1H, m, H3’), 1.24
(16H, m, H4’-11’), 1.15 (2H, m, H12’), 1.51 (1H, m, H13’), 0.86 (6H, d,
J = 6.6 Hz, H14’, 15), 13C NMR (CDCl3, 150 MHz) δ 166.3 (benzyl CO), 165.9
(benzyl CO), 133.14 (Ph), 133.09 (Ph), 130.0 (Ph), 129.9 (Ph), 129.8 (Ph),
129.8 (Ph), 129.7 (Ph), 129.7 (Ph), 128.4 (Ph), 128.4 (Ph), 128.4 (Ph), 128.4
(Ph), 71.9 (C-4), 71.1 (C-2), 69.0 (C-3), 63.7 (C-1), 39.1 (C-15), 30.0, 29.74,
29.69, 29.69, 29.61, 29.61, 29.5, 26.1 (C-7-14), 29.62 (C-5), 28.0 (C-16), 27.4
(C-6), 22.7 (C-17), 22.7 (C-18); HRESI/MS [M+Na]+ m/z 533.3227 (calcd for
C32H46O5Na, 533.3237).
Screening system for RNA 5ʹ-triphosphatase inhibitor
Nucleoside triphosphatase assay. The S. cerevisiae Cet1(201-549)p was
expressed in Escherichia coli as an N-terminal hexahistidine (His)-tagged
protein and purified by nickel-agarose and ion exchange chromatography.
An nucleoside triphosphatase assay with Cet1(201-549)p was performed on a
96-well microplate for initial screening of metabolites from actinomycetes.
Aliquots of 40 μl of a reaction mixture containing 50 mM Tris-HCl (pH 7.5),
2 mM MnCl2 and 8 nM Cet1(201-549)p were dispensed into each well, and
then 5 μl of test sample was added to the mixture. After preincubation for
5 min, the reaction was started by adding 5 μl of 400 μM ATP. After incubation
at 30 °C for 1 h, 75 μl of MicroMolar Phosphate Assay Reagent (ProFoldin,
Hudson, MA, USA) was added, and the mixtures were incubated for 5 min.
The absorbance at 620 nm was read using a plate reader (ARVOSX 1420
Multilabel counter; PerkinElmer, Waltham, MA, USA).
RNA 5ʹ-triphosphatase assays for Cet1p and hCap
The hCap1 was expressed in E. coli as an N-terminal His-tagged protein and
purified by nickel-agarose and ion exchange chromatography. RNA 5ʹ-tripho-
sphatase assays for Cet1p and hCap were performed as described previously
with a minor modification.24,25 The reaction mixtures for Cet1p containing
50 mM Tris-HCl (pH 7.9), 0.5 mM MgCl2, 40 μg of bovine serum albumin and
4 nM Cet1(201-549)p, and for hCap1 containing 50 mM Tris-HCl (pH 7.9),
2 mM DTT, 40 μg of bovine serum albumin and 0.23 nM hCap in a final
volume of 10 μl were preincubated with various compounds for 10 min. The
reaction was then started by adding 50 nM [γ32P]-radiolabeled triphosphate-
ended poly(A). After incubation at 30 °C for 10 min, the reaction products were
analyzed by polyethyleneimine cellulose TLC with 0.5 M potassium phosphate
buffer (pH 3.4). The TLC plate was exposed to an imaging plate and visualized
using the phosphorimager Typhoon Variable Model Imager (GE Healthcare,
Piscataway, NJ, USA), and the 50% inhibition concentration (IC50) was
calculated by four-parameter logistic curve fitting.
Methyl β- D-glucofuranosiduronic acid γ- lactone from 2 and
D-glucuronic acid (6 and 6ʹ)
The fraction No. 26 to 27 obtained by hydrophilic interaction liquid
chromatography described above was evaporated in vacuo to give 2.0 mg of
methyl-β-D-glucofuranosiduronic acid γ-lactone (6) as a colorless syrup:
1H NMR (600 MHz, CD3OD) δ 4.91 (1H, s, H1), 4.91 (1H, dd, J = 4.6,
6.5 Hz, H4), 4.80 (1H, d, J = 4.6 Hz, H3), 4.49 (1H, d, J = 6.4 Hz, H5), 4.18
(1H, s, H2), 3.33 (3H, s, OCH3). 13C NMR (CD3OD) δ 177.1 (C =O), 111.6
(C-1), 84.7 (C-3), 79.5 (C-4), 78.6 (C-2), 70.6 (C-5), 55.7 (OCH3); HRESI/MS
[M+Na]+ m/z 213.0372 (calcd for C7H10O6Na, 213.0370).
Antimicrobial activity
As an authentic sample of compound 6, 38.0 mg of D-glucuronic acid
MICs were determined by the standard agar dilution method recommended by
the Clinical Laboratory Standards Institute (CLSI) guidelines.26 Bacteria were
incubated on Mueller-Hinton agar (Becton Dickinson) at 37 °C for 18 h,
(Tokyo Chemical Industry Co. Ltd.) was dissolved in 3
chloride-methanol and stirred at room temperature for 1 h. The reaction
mixture was concentrated and the residue was applied for hydrophilic
M of hydrogen
interaction liquid chromatography using the same method described above. whereas yeast were incubated for 42 h.
The Journal of Antibiotics

Novel Cet1p inhibitors from actinomycete
M Igarashi et al
8
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ACKNOWLEDGEMENTS
This study was supported by the Japan Society for the Promotion of Science
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