Linfuranones B and C, Furanone-Containing Polyketides from a Plant-Associated Sphaerimonospora mesophila

Article abstract of DOI:10.1021/acs.jnatprod.8b00071

Two new furanone-containing polyketides, linfuranones B and C, were isolated from a plant-associated actinomycete of the genus Sphaerimonospora. Their structures were determined by NMR and MS spectroscopic analyses, and the absolute configurations were established by anisotropic methods and chemical degradation approaches. In silico analysis of biosynthetic genes suggested that linfuranone B is generated from linfuranone C by oxidative cleavage of the polyketide chain. Linfuranones B and C induced preadipocyte differentiation into matured adipocytes at 20-40 μM without showing cytotoxicity.

Full text of DOI:10.1021/acs.jnatprod.8b00071

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Linfuranones B and C, Furanone-Containing Polyketides from a
Plant-Associated Sphaerimonospora mesophila
Hirofumi Akiyama,† Chantra Indananda,‡ Arinthip Thamchaipenet,§ Atsuko Motojima,⊥
Tsutomu Oikawa,⊥ Hisayuki Komaki,∥ Akira Hosoyama,# Akane Kimura,# Naoya Oku,†
and Yasuhiro Igarashi*,†
†Biotechnology Research Center, Toyama Prefectural University, Imizu, Toyama 939-0398, Japan
‡Department of Biology, Faculty of Science, Burapha University, Chonburi 20131, Thailand
§Actinobacteria Research Unit, Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand
⊥Department of Nutritional Biochemistry, School of Nutrition and Dietetics, Kanagawa University of Human Services, Yokosuka,
Kanagawa 238-8522, Japan
∥Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Kisarazu, Chiba 292-0818, Japan
#NBRC, Shibuya, Tokyo 151-0066, Japan
S
* Supporting Information
ABSTRACT: Two new furanone-containing polyketides, linfuranones B and C, were isolated from a plant-associated
actinomycete of the genus Sphaerimonospora. Their structures were determined by NMR and MS spectroscopic analyses, and
the absolute configurations were established by anisotropic methods and chemical degradation approaches. In silico analysis of
biosynthetic genes suggested that linfuranone B is generated from linfuranone C by oxidative cleavage of the polyketide chain.
Linfuranones B and C induced preadipocyte differentiation into matured adipocytes at 20−40 μM without showing cytotoxicity.
acterial polyketides synthesized by modular polyketide
Bsynthases (type I PKSs) are one of the largest groups of
natural products. This group is characterized by its structural
variation and complexity arising from multiple chiral centers,
postmodification by tailoring enzymes, and/or coupling with
other pathways such as nonribosomal peptide synthesis.1
3(2H)-Furanone is a relatively minor structural element in
polyketides. To date, ca. 20 linear polyketides bearing a
furanone ring at their carboxy terminus have been found from
mollusks, fungi, actinomycetes, and myxobacteria (Figure 1).2
The actinomycete strain Sphaerimonospora mesophila
GMKU 363 was isolated from a root tissue of the Thai
medicinal herb “Lin Ngu Hoa” (Clinacanthus siamensis
Bremek.) collected in Chachoengsao Province, Thailand. We
previously reported the isolation of a furanone-containing
polyketide, linfuranone A (1),3 and then found its acylated
congener linfuranone B (2), which will be reported in this
paper. Accordingly, the draft genome of strain GMKU 363 was
sequenced to assess its biosynthetic potential in secondary
metabolism as well as to identify the biosynthetic genes for 1
and 2.4 A deduced biosynthetic gene cluster contains five PKS
genes in which a total of 11 modules are present along with the
genes of tailoring enzymes for furanone construction (Figure
11, Table 2).4 The PKS module organization predicted that the
intact PKS product is assembled from six methylmalonates and
five malonates (Figure 2). However, contrary to this
prediction, the main carbon chain of 1 and 2 is five carbons
shorter than that of the predicted structure. This inconsistency
between the predicted structure and the actually isolated
structures was suggestive of the presence of linfuranone
congeners other than 1 and 2, prompting us to reinvestigate
the metabolites of this strain.
Received: January 22, 2018
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00071
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Figure 1. Structures of linfuranones A−C (1−3) and related 3(2H)-furanones.
also from the COSY analysis. This fragment was connected to
C17 through C16 based on the HMBC correlations from H26
to C15, C16, and C17. Furthermore, the linear carbon chain
from C6 to C12, consisting of two fragments H6/H7/H8 and
H9/H10/H11, was established by the long-range correlations
from H11 to C12, H13 to C11, and H9 and H10 to C6. The
presence of a furanone substructure was deduced by the
HMBC correlations from the H24 methyl to the three sp2
carbons C3, C4, and C5 and the H1 methyl to C3 and
hemiketal carbon C2. The furanone moiety and the above-
defined chain structure were joined at C5 and C6 on the basis
of correlations from H6 to C4 and C5. The planar structure of
2 was thus determined as an O-acyl congener of 1 (Figure 3).
Absolute configurations at C7 and C15 of 2 were
determined by applying the modified Mosher’s method.5
MTPA esterification of 2 by the treatment with (S)- or (R)-
MTPACl gave tris(R)- and (S)-MTPA esters (4a and 4b).
Positive ΔδS−R values for H8 to H14 and H25 protons and
negative values for H6, H17, H18, H26, and H27 protons
indicated the 7R and 15R configurations (Figure 4). The
relative configuration at C14 was determined on the basis of
3JHH values and NOESY correlations. The large coupling
constant of 8.5 Hz between H14 and H15 indicated the anti
relationship of these protons around the C14−C15 axis
(Figure 5). C13 and C16 were also placed in an anti position
around the same axis on the basis of the NOESY correlations
for H13/H15, H14/H26, and H17/H25 (Figure 5). There-
fore, the relative configuration of the C25 methyl to 15-OH
was assigned anti, and thus the 14R configuration was
established. The remaining C18 configuration was determined
by chiral HPLC analysis of a lactic acid fragment obtained by
degradation of 2. Compound 2 was subjected to ozonolysis
followed by an oxidative workup with hydrogen peroxide. The
reaction mixture was then hydrolyzed in alkaline solution,
affording a mixture containing lactic acid derived from three
carbons, C17/C18/C19 (Figure 6). Chiral HPLC analysis in
comparison with the authentic (R)- and (S)-lactic acids
showed the occurrence of the (R)-enantiomer in the degraded
product mixture, thereby establishing the 18R configuration
(Figure 6).
RESULTS AND DISCUSSION
■
To search for a metabolite that has the aforementioned
predicted skeleton, culture conditions were examined and the
optimal growth temperature of strain GMKU 363 was found to
be around 37 to 40 °C, higher than the temperature commonly
used for secondary metabolite production by actinomycetes as
well as for the production of linfuranone A (1). The strain was
thus precultured and cultured at 37 °C for 4 days and 7 days,
respectively, and metabolites were analyzed in comparison with
those produced at 30 °C. In the culture extract obtained by
fermentation at 37 °C, a new intense peak that showed a
similar UV−vis spectrum to the furanone chromophore was
observed near the peak of linfuranone B (2) (Supporting
Information, Figure S17). This new peak was not detectable
when the strain was cultured at 30 °C. UV-guided purification
from the extract led to the isolation of another furanone-type
polyketide, linfuranone C (3), that has the same carbon
skeleton as the predicted structure (Figures 1 and 2). Herein,
we describe the isolation, structure elucidation, biological
property, and biosynthesis of 2 and 3.
Linfuranone B (2) was obtained as a pale yellow, amorphous
solid. The high-resolution ESITOFMS analysis gave a
molecular formula of C27H42O7, which was consistent with
1
the H and 13C NMR data. The IR spectrum indicated the
presence of hydroxy (3383 cm−1) and carbonyl (1695 and
1611 cm−1) groups. The UV spectrum with the absorption
maxima at 232 and 282 nm was closely similar to that of
linfuranone A,3 suggesting that 2 was a new congener. The 13
C
NMR and HSQC analysis confirmed the presence of 27
carbons attributable to three oxygenated sp2 carbons, seven
olefinic carbons (five are proton-bearing), one hemiketal
carbon, four sp3 methines (three are oxygenated), six sp3
methylenes, and six methyl carbons. Due to the epimerization
at the hemiketal carbon, two separated resonances were
observed for some of the protons and carbons (Table 1).2g−j
Analysis of the COSY spectrum allowed assignment of five
proton spin systems. First, contiguous correlations from H23
to H20, together with HMBC correlations from H20 and H21
to the carbonyl carbon C19 (δC 173.7), established an n-
pentanoyl group. The H18 methine had an HMBC correlation
to C19, and thereby the pentanoyl group was connected to
C18 through an ester linkage. A five-carbon fragment from
H12 to H15 with a methyl substituent on C14 was identified
Linfuranone C (3) was also obtained as a pale yellow,
amorphous solid. Its NMR, IR, and UV spectroscopic data
were similar to those for linfuranone B (2). The high-
B
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1
Table 1. H and 13C NMR Data for Linfuranones B (2) and C (3) in CDCl3
linfuranone B (2)
linfuranone C (3)
a
c
b
a
c
position
1
δC
δH mult (J in Hz)
HMBC
δC
δH mult (J in Hz)
HMBC
21.8
22.0
1.47, s
1.49, s
2, 3
2, 3
21.9
22.2
1.50, s
1.52, s
2, 3
2, 3
2
3
4
102.8
102.9
203.3
203.9
109.3
110.1
184.9
37.1
102.60
102.65
202.8
203.4
109.4
110.0
184.4
37.1
5
6
2.55, dd (14.1, 8.0)
2.75, m
2.46, dd (14.2, 2.8)
2.76, dd (14.2, 5.4)
4.09 m
4, 5, 7, 8
4, 5, 7, 8
4, 5, 7, 8
4, 5, 7, 8
5
2.60, dd (14.3, 8.1)
2.74, dd (14.3, 4.2)
2.49, m
2.75, dd (14.3, 9.5)
4.03, m
4, 5, 7, 8
4, 5, 7, 8
4, 5, 7, 8
4, 5, 7, 8
5
37.4
37.4
7
68.4
69.0
36.8
36.9
28.9
68.9
68.5
36.80
36.85
28.2
4.01 m
1.66 m
5
4.09, m
5
8
6, 7, 9, 10
1.66, m
7, 9, 10
9
2.20, m
7, 8, 11
8, 9, 12
9, 13
2.24, m
7, 8, 10, 11
8, 9, 12
28.9
10
11
12
13
132.3
132.4
131.06
131.12
131.9
132.0
134.2
134.3
40.9
81.26
81.27
138.4
128.6
67.8
5.62, dt (14.5, 7.1)
6.06, dd (14.5, 10.4)
6.11, dd (14.7, 10.4)
5.48, dd (14.7, 8.3)
132.4
132.5
131.09
131.15
132.08
132.14
134.1
134.2
41.2
5.63, dt (14.4, 6.8)
6.06, dd (14.4, 10.4)
6.11, dd (14.3, 10.4)
5.47, dd (14.3, 8.7)
9, 13
10, 11, 14
10, 11, 14
11, 14, 15, 25
11, 14, 15, 25
14
15
2.30, m
3.63, d (8.5)
25
2.34, m
3.69, d (8.1)
12, 13, 15, 25
13, 14, 16, 17, 25,
26
13, 14, 16, 17,
25, 26
81.3
16
17
18
138.0
127.6
45.96
46.00
212.7
212.8
40.8
5.35, d (8.5)
5.57, dq (8.5, 6.0)
15, 26, 27
16, 17, 19, 27
5.31, d (9.3)
3.43, m
15, 18, 19, 26, 27
16, 17, 19, 27
19
20
173.7
34.5
2.27, t (7.5)
19, 21, 22
2.44, m
19, 21, 22
40.9
21
22
23
24
27.2
22.4
13.9
5.9
1.59, tt (7.5, 7.5)
1.34, tq (7.5, 7.5)
0.91, t (7.5)
19, 20, 22, 23
20, 21, 23
20, 21, 22
3, 4, 5
26.1
22.6
14.1
5.9
1.52, m
19, 20, 22, 23
20, 21, 23
21, 22
1.28, tq (7.4, 7.4)
0.90, t (7.4)
1.66, s
1.67, s
3, 4, 5
1.67, s
25
26
17.2
11.6
0.86, d (6.5)
1.69, s
13, 14, 15
15, 16, 17
17.4
12.0
12.1
16.78
16.82
0.91, d (6.5)
1.680, s
1.684, s
1.129, d (6.8)
1.133, d (6.8)
13, 14, 15
15, 16, 17
15, 16, 17
17, 18
27
20.7
1.26, d (6.0)
17, 18
17, 18
a
b
c
Recorded at 500 MHz. Recorded at 125 MHz. HMBC correlations are from proton(s) stated to the indicated carbon.
resolution ESITOFMS analysis gave the molecular formula
C27H42O6, which indicated a loss of one oxygen atom from 2.
The presence of an n-pentanoyl group was detected in the
COSY spectrum, but the ester carbonyl carbon was missing in
the 13C NMR spectrum, and the methylene protons H20 and
H21 of the n-pentanoyl group and H27 doublet methyl
protons displayed HMBC correlations to a keto carbon at δC
212.8 (C19). This doublet methyl was correlated to a methine
(δH 3.44; δC 46.1) that showed a COSY correlation to olefinic
methine H17 and a long-range correlation to C19. These
correlation data indicated that the ester oxygen in 2 was
missing in 3, where the n-pentanoyl group was directly
connected to C14. Further NMR analysis confirmed that the
remaining part from C1 to C15 was the same as the
corresponding part of 2 (Figure 7, Table 1). Thus, the planar
structure of 3 was determined as shown in Figure 5. The
carbon skeleton of 3 was identical with that of the structure
C
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Table 2. Annotated Putative ORFs in the Biosynthetic Gene Cluster of Linfuranones
protein homologue whose function is characterized
size
(aa)
a
orf6- accession number
proposed function
description, origin, accession number
%
134
133
132
131
WP_055480424
WP_055480423
WP_055480220
WP_055480219
3083 PKS
4795 PKS
3527 PKS
5362 PKS
type I polyketide synthase (cmiP4), Streptomyces sp. MJ635-86F5, BAO66529
modular polyketide synthase (Nmd1), Streptomyces sp. RK95-74, BAW35608
modular polyketide synthase (Nmd6), Streptomyces sp. RK95-74, BAW35613
type I modular polyketide synthase (PkeAIII), Saccharopolyspora erythraea NRRL 56/66
2338, AAQ94248
53/64
50/61
56/66
130
129
WP_055480218
WP_063842922
1510 PKS
type I PKS (HcaA), Streptomyces hygroscopicus, CCF23200
51/60
52/66
488
FAD-dependent
monooxygenase
Baeyer−Villiger monooxygenase (ORF 8), Streptomyces aculeolatus NRRL 18422,
ABB88524
128
WP_055480421
840
cytochrome P450
hydroxylase
monooxygenase AufB, Stigmastella aurantiaca DW4/3−1, CAO98846
56/70
127
126
WP_055480217
WP_055480216
250
545
type II thioesterase
FAD-dependent
monooxygenase
type II thioesterase, Streptomyces sp. NBRC 110027, GAO07626
phenylacetone monooxygenase (PamO), Thermobifida fusca YX, AAZ55526
53/70
57/69
a
Identity/similarity.
for H6, H14 to H18, and H25 to H27 were negative,
establishing 7R and 15R configurations (Figure 8). Similar to
the case of 2, the relative configuration of the methyl group at
C14 was assigned anti to the 15-OH group by NOESY analysis
and a coupling constant.
The remaining chirality at C18 of 3 was determined through
chemical degradation. In order to avoid epimerization at C18,
the C19 keto group was reduced to a hydroxy group by L-
selectride. Then, the C16−C17 double bond was cleaved by
ozonolysis and following oxidative workup gave a mixture
containing α-methyl-β-hydroxyheptanoic acid (nat-6) derived
from the C17−C23 portion of 3 (Figure 9). This reaction
mixture was subjected to (R)-PGME (phenylglycine methyl
ester)7 derivatization to obtain the corresponding amide,
which was compared with the (R)-PGME amides of four
possible synthetic stereoisomers of 6 by LC/MS (Figures 9
and 10). The (R)-PGME derivative prepared from degradation
of 3 gave two peaks, corresponding to (2S,3R)- and (2S,3S)-6
at 19.1 and 23.1 min, respectively, in a ratio of ca. 1:3.
Therefore, the S configuration of the methyl group in 6 derived
from 3 and thereby the 18R configuration for 3 were
established.
Genome analysis of S. mesophila GMKU 363 identified three
type I PKS gene clusters,4 one of which was assigned to that for
the linfuranone biosynthesis based on in silico analysis of PKS
module/domain organizations (Figure 11). This type I PKS
system comprises five open reading frames (orf6−134, orf6−
133, orf6−132, orf6−131, orf6−130) encoding 11 modules.
Module 1 was deduced as a loading module since the active
site cysteine of the ketosynthase (KS) domain was replaced
with glutamine.8 Analysis of the signature amino-acid residues
of AT domains predicted that AT domains in modules 1, 3, 4,
5, 10, and 11 have specificity to methylmalonyl-CoA and those
Figure 2. Structure of linfuranone skeleton predicted by in silico
genomic analysis. See Figure 11 for biosynthetic gene organization.
predicted by in silico genomic analysis of the PKS gene cluster
of linfuranones.
The absolute configurations at C7 and C15 were elucidated
by chiral anisotropic analysis. Compound 3 was derivatized to
MPA (α-methoxyphenylacetic acid) esters6 by treatment with
(R)- and (S)-MPA to give tris(R)- and (S)-MPA esters (5a
and 5b), respectively. The signs of ΔδR−S values for the
protons from H7 to H14 and H25 were positive, while those
Figure 3. COSY and key HMBC correlations for linfuranone B (2).
D
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Figure 4. ΔδS−R values for tris-MTPA derivatives (4a and 4b) of 2.
enzymes are encoded in the linfuranone biosynthetic gene
locus: two FAD-dependent monooxygenases (orf6−129, orf6−
126) and a cytochrome P450 hydroxylase (orf6−128). The
monooxygenase ORF6-129 has significant homology to
Baeyer−Villiger monooxygenases ORF 8 (52% identity and
66% similarity), ORF 13 (48% identity and 58% similarity),
and AufJ (47% identity and 61% similarity) responsible for
furanone formation in the biosynthesis of E-837, E-492, and
aurafurons, respectively.2g,12 The hydroxylase ORF6-128
shows high homology to AufB (56% identity and 70%
similarity) responsible for hydroxylation of the furanone unit
in aurafuron biosynthesis.12 ORF6-128 also displays high
homology to the hydroxylases in the biosynthesis of E-837 and
E-492.2g Another FAD-dependent monooxygenase, ORF6-126,
shares high homology with phenylacetone monooxygease
PamO from a thermophilic bacterium, Thermobifida fusca
(57% identity and 69% similarity).13 PamO has the ability to
catalyze the Baeyer−Villiger oxidation of cyclic and acyclic
ketones such as phenylacetone. Based on these in silico
investigations, ORF6-126 is proposed to catalyze the oxygen
insertion between C18 and C19 of linfuranone C (3) that gives
rise to linfuranone B (2), whereas ORF6-129 and ORF6-128
are most likely involved in furanone formation (Figures 11 and
12). Orf6-127 flanked by the oxidative tailoring enzymes in the
biosynthetic gene cluster encodes a type II thioesterase (Table
2) which likely plays a roll of an editing enzyme that catalyzes
the removal of aberrant polyketide intermediates inappropri-
ately synthesized by PKSs.14 As there is no hydrolytic enzyme
encoded in or near the cluster, hydrolysis of 2 to 1 might be a
nonenzymatic process.
Figure 5. Relative configuration of C14 and C15 of 2 determined by
NOEs.
As linfuranone A (1) has the ability to induce differentiation
of murine ST-13 preadipocytes into mature adipocytes, effects
of linfuranones B (2) and C (3) on the same cell lines were
examined. By the treatment with 20 μM of 2 and 3, 22% and
45% of preadipocytes were differentiated, respectively. Both
compounds were more potent than 1 (9% differentiation at 20
μM). At the concentration of 40 μM, 33% and 73% of
preadipocytes were differentiated into adipocytes by treatment
with 2 and 3, respectively. 2 and 3 were inactive against
Escherichia coli, Micrococcus luteus, and Candida albicans at 25
μg/mL.
Figure 6. Chiral HPLC analysis of degraded product of 2.
in modules 2, 6, 7, 8, and 9 likely incorporate the malonyl
extender unit.9 The structures of linfuranones suggested that
the ketoreductase (KR) domain in module 3 and the
dehydratase (DH) domain in module 9 are inactive, also
being predicted by the sequence analysis that KR3 lacks active
residues LDD and W,10 and DH9 lacks hallmark motifs
GYxYGPxF and LPFxW.11 Three potential post-PKS tailoring
Figure 7. COSY and key HMBC correlations for linfuranone C (3).
E
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Figure 8. ΔδR−S values for tris-MPA derivatives (5a and 5b) of 3.
Figure 9. Degradation of 3 and PGME derivatization.
example in which Baeyer−Villiger-type oxidation is involved in
the oxygen insertion into a linear polyketide chain.15
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a JASCO DIP-3000 polarimeter. UV spectra were
recorded on a Hitachi U-3210 spectrophotometer. IR spectra were
measured on a PerkinElmer Spectrum 100. NMR spectra were
obtained on a Bruker AVANCE 500 spectrometer in CDCl3 using the
signals of the residual solvent protons (δH 7.27) and carbons (δC
77.0) as internal standards. HR-ESITOFMS were recorded on a
Bruker micrOTOF focus.
Microorganism. Strain GMKU 363 was isolated from a root of
the Thai medicinal plant “Lin Ngu Hao” (Clinacanthus siamensis
Bremek.) collected in Chachoengsao Province, Thailand. It was
initially identified as a member of the genus Microbispora in our
previous study.3 However, this strain was reassigned to Sphaer-
imonospora mesophila16 on the basis of genome sequence-based
classification17 in comparison with Sphaerimonospora mesophila
NBRC 14179T (BCRI00000000.1).
Figure 10. LC/MS analysis of the (R)-PGME derivative of the
degraded product of 3.
Fermentation. Strain GMKU 363 cultured on a Bn-2 agar
medium consisting of 0.5% soluble starch, 0.5% glucose, 0.1% meat
extract (Kyokuto Pharmaceutical Industrial Co., Ltd.), 0.1% yeast
extract (Difco Laboratories), 0.2% NZ-case (Wako Pure Chemical
Industries, Ltd.), 0.2% NaCl, 0.1% CaCO3, and 1.5% agar was
inoculated into 500 mL K-1 flasks each containing 100 mL of the V-
22 seed medium (pH 7.0) consisting of 1% soluble starch, 0.5%
glucose, 0.3% NZ-case, 0.2% yeast extract, 0.5% Tryptone (Difco
Laboratories), 0.1% K2HPO4, 0.05% MgSO4·7H2O, and 0.3% CaCO3.
The flasks were placed on a rotary shaker (200 rpm) at 37 °C for 4
days. The seed culture (5−10%) was transferred into 500 mL K-1
flasks each containing 100 mL of the A-11 M production medium
In conclusion, two new furanone-containing polyketides,
linfuranones B (2) and C (3), were isolated from an
actinomycete of the genus Sphaerimonospora. Compound 3
was discovered through in silico analysis of biosynthetic genes
and temperature optimization for production fermentation.
Compound 2 is generated from 3 by oxidative cleavage of the
polyketide backbone by Baeyer−Villiger oxidation. This type
of decoration of the polyketide main carbon chain is not
common. DTX-4, a dinoflagellate metabolite, is a preceding
F
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Figure 11. Putative biosynthetic gene cluster of linfuranones.
Figure 12. Plausible biosynthetic relationship among linfuranones A−C.
(pH 7.0) consisting of 2.5% soluble starch, 0.2% glucose, 0.5% yeast
extract, 0.5% polypeptone (Wako Pure Chemical Industries, Ltd.),
0.5% NZ-amine (Wako Pure Chemical Industries, Ltd.), 0.3%
CaCO3, and 1% Diaion HP-20 (Mitsubishi Chemical Co.). The
inoculated flasks were placed on a rotary shaker (200 rpm) at 37 °C
for 7 days.
Nacalai Tesque) HPLC using an isocratic MeCN/H2O (50:50) at 4
mL/min, yielding linfuranone B (2, 141 mg, tR 20.3 min) and
linfuranone C (3, 22 mg, tR 15.6 min) as a pale yellow, amorphous
solid.
Linfuranone B (2): pale yellow, amorphous solid; [α]22D +77 (c 1.0,
MeOH); UV (MeCN) λmax (log ε) 232 (5.55), 282 (5.04); IR νmax
3383, 1695, 1611 cm−1; 1H and 13C NMR data, see Table 1;
HRESITOFMS [M − H]− 477.2859 (calcd for C27H41O7, 477.2858).
Linfuranone C (3): pale yellow, amorphous solid; [α]22D −67 (c 0.
40, MeOH); UV (MeCN) λm1ax (log ε) 227 (4.86), 281 (4.87); IR
νmax 3383, 1695, 1611 cm−1; H and 13C NMR data, see Table 1;
HRESITOFMS [M − H]− 461.2905 (calcd for C27H41O6, 461.2909).
Tris(R)-MTPA ester of 2 (4a). To a solution of 2 (2.0 mg, 4.2 μmol)
in dry pyridine (100 μL) was added (S)-MTPA chloride (7 μL, 37
μmol) at room temperature. After standing for 1 h, the reaction
mixture was concentrated under reduced pressure, and the residue
was purified by silica gel column chromatography (n-hexane/EtOAc,
Extraction and Isolation. At the end of the fermentation period,
50 mL of 1-butanol was added to each flask, and the flasks were
allowed to shake for 1 h. The mixture was centrifuged at 6000 rpm for
10 min, and the organic layer was separated from the aqueous layer
containing the mycelium. Evaporation of the solvent gave
approximately 7.8 g of crude extract from 10 L of culture. The
crude extract (7.8 g) was subjected to silica gel column
chromatography with a step gradient of CHCl3−MeOH (1:0, 20:1,
10:1, 4:1, 2:1, 1:1, and 0:1 v/v). Fraction 4 (4:1) was concentrated to
provide 2.2 g of brown oil, which was further purified by reversed-
phase ODS column chromatography with a gradient of MeCN−H2O
(2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 v/v). Fraction 5 (6:4) was
evaporated, and the remaining aqueous solution was extracted with
EtOAc. After drying with anhydrous Na2SO4, the organic layer was
concentrated to give semipure linfuranones. Final purification was
achieved by preparative ODS (Cosmosil C18-AR-II, 10 × 250 mm,
1
8:1−1:1) to give tris(R)-MTPA ester 4a (2.8 mg, 60% yield): H
NMR (500 MHz, CDCl3) δ 0.88 (H25), 0.89 (H23), 1.27 (H27),
1.32 (H22), 1.48 (H1), 1.50 (H1), 1.57 (H21), 1.73 (H26), 1.74
(H24), 1.75 (H24), 1.77 (H8), 1.99 (H9), 2.26 (H20), 2.50 (H14),
2.79 (H6), 3.01 (H6), 5.35 (H13), 5.51 (H17), 5.58 (H18), 5.81
G
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(H11), 5.87 (H12); HR-ESITOFMS m/z 1165.3756 [M + K]+ (calcd
for C57H63F9O13K 1165.3757).
Tris(S)-MTPA ester of 2 (4b). In the same manner as described for
evaporated to dryness under reduced pressure. The residue was
subjected to (R)-PGME amidation and the following LC/MS analysis.
LC/MS analysis of the degradation product of 3. The (R)-PGME
derivative of 3-hydroxy-2-methylheptanoic acid (6) derived from the
degradation of 3 was compared with the (R)-PGME derivatives of
four possible stereoisomers of 6 prepared by chemical synthesis (see
Supporting Information). LC/MS analysis was performed on an
Agilent HP1200 system and Bruker micrOTOF Focus using an ODS
column (COSMOSIL 5C18-AR-II, 2.0 × 150 mm) at a flow rate of 0.3
mL/min with an isocratic elution (40% MeCN in distilled water).
Retention times of the standard samples were 19.3 min for (2S,3R)-6,
20.0 min for (2R,3S)-6, 23.1 min for (2S,3S)-6, and 25.1 min for
(2R,3R)-6. Under the same conditions, the (R)-PGME derivative of
the degraded product of 3 gave two peaks, at 19.1 and 23.1 min,
corresponding to (2S,3R)- and (2S,3S)-isomers, respectively.
Adipocyte Differentiation Assay. The assay was carried out
according to the reported procedure.18 In brief, mouse-derived ST-13
preadipocytes were plated at 1 × 104 cells/mL in 24-well dishes
containing 1 mL of a basal medium containing DME/F-12, 10% fetal
bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin
on day 1 and cultured at 37 °C in a humidified atmosphere containing
5% CO2. Moreover, the fresh basal medium containing the indicated
concentrations of linfuranones (or 20 nM rosiglitazone (positive
control)) was replaced on day 2, 5, and 8. On day 12, the ST-13 cells
were washed three times with phosphate-buffered saline, fixed with
10% formalin neutral buffer solution at room temperature for 10 min,
and then washed with distilled water to remove formalin solution.
Furthermore, the cells were rinsed with 60% 2-propanol for 5 min,
stained with 0.24% Oil Red O at room temperature for 20 min, and
then photographed under a phase contrast microscope (100×
magnification) equipped a CCD camera (Leica Microsystems Japan,
Tokyo, Japan).
4a, 4b (2.7 mg, 55% yield) was prepared from 2 (2.0 mg) and (R)-
1
MTPA chloride: H NMR (500 MHz, CDCl3) δ 0.89 (H25), 0.91
(H23), 1.25 (H27), 1.35 (H22), 1.51 (H26), 1.57 (H21), 1.61 (H1),
1.68 (H1), 1.78 (H8), 1.83 (H24), 1.84 (H24), 2.13 (H9), 2.28
(H20), 2.55 (H14), 2.71 (H6), 2.92 (H6), 5.44 (H13), 5.50 (H17),
5.55 (H18), 5.95 (H11), 6.02 (H12); HR-ESITOFMS m/z
1149.4010 [M + Na]+ (calcd for C57H63F9O13Na 1149.4017).
Alkaline hydrolysis of 2 to linfuranone A (1). Compound 2 (5.0
mg, 10 μmol) was dissolved in 5% NaOMe/MeOH (2.6 mL) and
stirred for 30 min at room temperature. The reaction mixture was
neutralized by adding 5% HCO2H solution (4 mL) and then
evaporated to dryness. The crude material was purified by HPLC
using a Cosmosil C18-AR-II column (10 × 250 mm) with an isocratic
solvent system of 30% MeCN in distilled water at a flow rate of 4.0
mL/min, monitoring at 280 nm to yield 1 (2.3 mg, tR = 12.1 min):
1
[α]23 −3.0 (c 0.10, MeOH); H and 13C NMR data matched those
D
of naturally occurring linfuranone A (1); HR-ESITOFMS m/z
393.2285 [M − H]− (calcd for C22H33O6 393.2283).
Oxidative Degradation of 2 and Chiral HPLC Analysis. To a
solution of 2 (2.0 mg, 4.2 μmol) in EtOAc (5 mL) was bubbled O3 at
−78 °C until the solution turned pale blue. After bubbling oxygen
into the solution at the same temperature for 15 min, HCO2H (0.75
mL) and 30% H2O2 (0.44 mL) were added, and the reaction mixture
was heated at 60 °C for 15 h. Then, the reaction mixture was
evaporated under vacuum, the resultant material was dissolved in
MeOH (200 μL), and 28% NaOMe solution (150 μL) was added.
After standing for 18 h at ambient temperature, the reaction mixture
was analyzed on a chiral HPLC (Sumichiral OA-5000, 4.6 × 150 mm;
1 mM CuSO4 solution; 1.0 mL/min; detection at 254 nm). Retention
times of the standard samples were 21.8 min for (S)-lactic acid and
27.8 min for (R)-lactic acid. Co-injection of the degraded product of 2
gave two peaks when injected with (S)-lactic acid and a single peak
when injected with (R)-lactic acid.
Tris(R)-MPA ester of 3 (5a). To a solution of 3 (1.0 mg, 2.2 μmol)
in dry CH2Cl2 (120 μL) were added (R)-MPA (1.5 mg, 9.0 μmol),
N,N-dimethyl-4-aminopyridine (DMAP, trace amount), and a
solution of diisopropylcarbodiimide (DIC, 1 mg, 7.9 μmol) in dry
CH2Cl2 (20 μL) at room temperature. After standing for 30 min, the
reaction mixture was diluted with ice/water and extracted with
EtOAc. The organic layer was concentrated under reduced pressure,
and the residue was purified by HPLC (n-hexane/EtOAc, 8:1−1:1) to
give tris(R)-MPA ester 5a (0.6 mg, 30% yield): 1H NMR (500 MHz,
CDCl3) δ 0.88 (H23), 0.90 (H25), 0.93 (H27), 1.22 (H22), 1.43
(H21), 1.70 (H8), 1.98 (H9), 2.22 (H20), 2.49 (H14), 2.72 (H6),
3.16 (H18), 5.06 (H17), 5.40 (H13), 5.47 (H10), 5.85 (H11), 5.96
(H12); HR-ESITOFMS m/z 929.4445 [M + Na]+ (calcd for
C54H66O12Na 929.4446).
Tris(S)-MPA ester of 3 (5b). In the same manner as described for
5a, 5b (0.5 mg, 25% yield) was prepared from 3 (1.0 mg, 2.2 μmol)
and (S)-MPA: 1H NMR (500 MHz, CDCl3) δ 0.78 (H25), 0.88
(H23), 1.05 (H27), 1.23 (H22), 1.46 (H21), 1.54 (H8), 1.66 (H9),
2.29 (H20), 2.44 (H14), 2.76 (H6), 3.31 (H18), 5.19 (H13), 5.23
(H10), 5.29 (H17), 5.52 (H11), 5.74 (H12); HR-ESITOFMS m/z
929.4447 [M + Na]+ (calcd for C54H66O12Na 929.4446).
Degradation of 3. To a stirred solution of 3 (0.25 mg, 0.5 μmol)
in dry THF (50 μL) was added L-selectride (1.0 M THF solution, 10
μL, 0.5 μmol) at 0−5 °C. After 1 h, L-selectride (40 μL, 0.5 μmol) was
further added to the solution, which was then stirred for 18 h at
ambient temperature. The reaction was quenched with a saturated
NH4Cl solution and extracted with EtOAc. The organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The residual material was dissolved in EtOAc (5 mL), to
which was bubbled O3 at −78 °C for 30 min. Then, formic acid (0.75
mL) and 30% H2O2 solution (0.40 mL) were successively added to
the reaction mixture, and the temperature was gradually raised to 60
°C. After stirring for 15 h at the same temperature, the mixture was
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.8b00071.
Experimental procedures and 1D and 2D NMR spectra
of compounds 2 and 3 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@
pu-toyama.ac.jp.
■
ORCID
Yasuhiro Igarashi: 0000-0001-5114-1389
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by JSPS KAKENHI Grant Number
24580156 to Y.I., Core-to Core Program, JSPS, and National
Research Council of Thailand (NRCT) to A.T.
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