Production of novel bispyrone metabolites in the cellular slime mold Dictyostelium giganteum induced by zinc(II) ion

Article abstract of DOI:10.1016/j.tet.2016.12.040

In this study, Zn²⁺induced the production of two new bispyrone-type metabolites, dictyobispyrone B and E, in the cellular slime mold Dictyostelium giganteum. Their structures were proposed on the basis of spectroscopic analysis and confirmed by chemical synthesis. They possess a novel α,α-bispyrone skeleton modified with long carbon chains. Both could be formed from two different polyketide chains through a novel biosynthetic pathway. Our results indicate that cultivation of cellular slime molds in the presence of Zn²⁺is a useful technique for discovering other structurally unique compounds.

Full text of DOI:10.1016/j.tet.2016.12.040

Accepted Manuscript
Production of novel bispyrone metabolites in the cellular slime mold Dictyostelium
giganteum induced by zinc(II) ion
Van Hai Nguyen, Haruhisa Kikuchi, Hiraku Sasaki, Kyoichi Iizumi, Yuzuru Kubohara,
Yoshiteru Oshima
PII:
S0040-4020(16)31326-6
10.1016/j.tet.2016.12.040
TET 28332
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 16 November 2016
Revised Date: 17 December 2016
Accepted Date: 19 December 2016
Please cite this article as: Nguyen VH, Kikuchi H, Sasaki H, Iizumi K, Kubohara Y, Oshima Y, Production
of novel bispyrone metabolites in the cellular slime mold Dictyostelium giganteum induced by zinc(II) ion,
Tetrahedron (2017), doi: 10.1016/j.tet.2016.12.040.
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ACCEPTED MANUSCRIPT
Production of Novel Bispyrone Metabolites in the Cellular
Slime Mold Dictyostelium giganteum Induced by Zinc(II) Ion
Van Hai Nguyen,a Haruhisa Kikuchi,a,* Hiraku Sasaki,b Kyoichi Iizumi,b Yuzuru Kubohara,b
Yoshiteru Oshima a,*
a Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku, Sendai
980-8578, Japan
b Graduate School of Health and Sports Science, Juntendo University, 1-1 Hiraga-gakuendai, Inzai,
Chiba 270-1695, Japan
*To whom correspondence should be addressed. Phone: +81-22-795-6824. Fax: +81-22-795-6821.
E-mail: hal@mail.pharm.tohoku.ac.jp (H.K.), oshima@mail.pharm.tohoku.ac.jp (Y.O.)
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Graphical Abstract
Production of Novel Bispyrone Metabolites in the Cellular Slime Mold Dictyostelium
giganteum Induced by Zinc(II) Ion
Van Hai Nguyen, Haruhisa Kikuchi,* Hiraku Sasaki, Kyoichi Iizumi, Yuzuru Kubohara, Yoshiteru
Oshima *
O
O
Zn2+
10
Dictyobispyrone B
O
O
O
O
O
Cultivation in the
presence of Zinc(II) ion
10
Cellular slime mold
Dictyostelium giganteum
Dictyobispyrone E
O
2

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Abstract
In this study, Zn2+ induced the production of two new bispyrone-type metabolites,
dictyobispyrone B and E, in the cellular slime mold Dictyostelium giganteum. Their structures were
proposed on the basis of spectroscopic analysis and confirmed by chemical synthesis. They possess
a novel α,α-bispyrone skeleton modified with long carbon chains. Both could be formed from two
different polyketide chains through a novel biosynthetic pathway. Our results indicate that
cultivation of cellular slime molds in the presence of Zn2+ is a useful technique for discovering
other structurally unique compounds.
Keyword
Cellular slime molds; Dictyostelium; Bispyrone; Structural elucidation
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1. Introduction
Cellular slime molds are well-studied organisms that are excellent models for investigating cell
and developmental biology because of their simple development patterns.1 Recently, our group has
been focusing on the utility of cellular slime molds as a source of natural compounds and has
isolated several structurally unique, biologically significant compounds from these molds.2–4
Dictyotriphenyl from Dictyostelium polycephalum is one of the first-discovered natural nitrogen-
containing m-terphenyls.4 Ppc-1 is a prenylated aromatic compound isolated from Polysphondylium
pseudo-candidum that has potent immunosuppressive effects in vitro and in vivo.5,6 The above
results show that cellular slime molds are an important source of lead compounds for medical
research.
In 2005, the genome sequencing of the cellular slime mold Dictyostelium discoideum clearly
revealed that it has approximately 45 polyketide synthase (PKS) gene clusters.7 Another species of
a cellular slime mold, Dictyostelium purpureum, has also been reported to exhibit 50 predicted PKS
genes.8 These numbers are even greater than those of Streptomyces avermitilis, which is known to
contain abundant secondary metabolites. Despite their potential, only a few secondary metabolites
from cellular slime molds have been reported thus far. Several gene clusters have been reported to
be transcriptionally suppressed under standard laboratory culture conditions.9 Thus, an improved
method for activating silent gene clusters is needed to obtain new compounds from cellular slime
molds.
On the other hand, metal ions have been reported to enhance the production of metabolites in
fungi. Cuero et al. reported that the treatment of Aspergillus flavus with the mineral ions Zn2+, Cu2+,
or Fe2+ induces changes in the mRNA levels as well as biosynthesis of aflatoxin and its precursor.10
Similarly, Ca2+, Cu2+, and Al3+ have been reported to be effective at enhancing the production of
palmarumycins C12 and C13 in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12.11
These studies demonstrate that the cultivation of fungi in the presence of added metal ions is a
promising process for obtaining more metabolites. One study reported that Zn2+ exhibits a specific,
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striking effect on the cell differentiation of D. discoideum.12 A subsequent work stimulated the
production of polyketides in a liquid culture of D. discoideum using Zn2+ or Cd2+ and thus found
that some of these polyketides are obtainable only by cultivation in the presence of each metal ion.13
These results suggest that Zn2+ can be utilized as a factor for stimulating metabolite production in
cellular slime molds.
To obtain novel bioactive compounds from cellular slime molds, we conducted screening to
evaluate the effect of Zn2+ on metabolite production in various species of cellular slime molds. We
found that Zn2+ enhances the production of two novel bispyrone-type compounds, dictyobispyrone
B (1) and E (2), in Dictyostelium giganteum (Figure 1).
Figure 1. Compounds isolated from Dictyostelium giganteum.
2. Results and discussion
We performed small-scale screening of 20 species of cellular slime molds. Each species was
cultured in an agar medium with or without Zn2+. To evaluate the effect of Zn2+, metabolites soluble
in ethyl acetate were separated by reversed-phase high-performance liquid chromatography (RP-
HPLC) and compared with the control sample. We found that the presence of Zn2+ does not
substantially affect metabolite production in D. discoideum or some other species, but it induces the
production of some metabolites in D. giganteum. Figure 2 reveals enhanced production of 1 and 2
relative to that under control conditions during the cultivation of D. giganteum in the presence of
0.5 mM Zn2+. We also found that 2 mM and 8 mM of Zn2+ inhibited the growth of D. giganteum,
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and decreased the amount of metabolites. Hence, we conducted the large-scale cultivation of D.
giganteum in the presence of 0.5 mM Zn2+. The fraction soluble in ethyl acetate was separated
multiple times by silica-gel column chromatography to afford an inseparable mixture. When the
mixture was separated by RP-HPLC, two peaks with retention times consistent with those of 1 and
2 were observed (see Supplementary Information).
Figure 2. Results of HPLC analysis of the ethyl acetate soluble extract of Dictyostelium giganteum.
The 1H and 13C NMR spectra of 1 and 2 suggest that they have common cyclic structures, each
with a different carbon chain. The common peaks in the 1H and 13C NMR spectra indicate two ester
carbonyl, four olefinic, oxymethine, methylene, and methyl carbons in the common structural unit.
1H–1H COSY and HMBC correlations of these groups confirm the partial structure unit I (Figure 3).
13
The chemical shift of C-10 at δC 177.9 in the C NMR spectrum suggests that C-10 is possibly
connected to O-9 and thus forms a pyrone ring. Therefore, C-2 and C-3 are possibly connected and
are part of another pyrone ring. Accordingly, the common structural unit of 1 and 2 was determined
to be bispyrone ring II (Figure 3).
1
13
As can be observed in the H and C NMR spectra, 1 has a double bond in its carbon chain.
From the molecular ion peak m/z 360.2301 [M]+ (calcd for C22H32O4 360.2299) observed by
HREIMS, we propose that the carbon chain of 1 is III in Figure 3. The E configuration was
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assigned to the C-11′–C-12′ double bond by comparison of the chemical shifts of C-10′ (δC 32.6)
and C-13′ (δC 17.9) with the corresponding calculated values (δC (E) 33.0, (Z) 27.0 for C-10′; δC (E)
17.0, (Z) 11.0 for C-13′). On the other hand, the carbon chain of 2 was proposed to be IV on the
basis of the molecular ion peak 348.2293 [M]+ (calcd for C21H32O4 348.2299) observed in HREIMS.
Finally, the connection between II and III or II and IV was established from the correlation peaks
for H-1′ or H-1′′ to C-10 in the HMBC spectrum. Accordingly, the planar structures of 1 and 2
were determined to be dictyobispyrones B and E, respectively.
Figure 3. Key 1H–1H COSY correlations (bold lines), key HMBC correlations (arrows), and partial
structures for the mixture of dictyobispyrone B (1) and E (2).
A mixture of the known compound dictyopyrone B (3)14 and a new compound, dictyopyrone E
(4), was also isolated from the ethyl acetate soluble extract of D. giganteum. Structural elucidation
of 3 and 4 by spectroscopic analysis was conducted in the same manner as that conducted for 1 and
2. We also performed the total synthesis of 4 to confirm its structure (see Supplementary
Information).
Although compound 1 and 2 could be separated by RP-HPLC, the amount of individual
compound was not enough for spectroscopic analysis except mass spectrometry experiments.
Therefore, elucidating the absolute stereochemistry at C-6 of them was difficult. A previous study
isolated dictyopyrones, a series of α-pyronoids having a pyrone ring modified with a long carbon
chain, from cellular slime molds.14 The structural similarity of the pyrone ring between
dictyobispyrones and dictyopyrones suggests that they exhibit the same absolute stereochemistry at
C-6. To test the hypothesis that the absolute stereochemistry of 1 and 2 at C-6 is S, we performed
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their total synthesis. Compound 1 was synthesized from the commercially available compounds (S)-
propylene oxide (5) and ethyl propiolate (6) (Scheme 1). Nucleophilic addition of ethyl propiolate
to propylene oxide15 and subsequent protection of the secondary alcohol by the methoxymethyl
group afforded compound 7. The condensation of 7 and β-ketoester 816 produced pyrone compound
9.17 Finally, 1 was formed by treatment of 9 with p-toluenesulfonic acid. Compound 2 could be
synthesized in the same manner as 1 by using β-ketoester 10 instead of 8.
Scheme 1. Total synthesis of dictyobispyrone B (1) and E (2).
The 1H and 13C NMR spectra of the isolated natural mixture of 1 and 2 were compared with the
combined spectrum of synthetic 1 and 2. Their agreement indicates that the proposed planar
structures of 1 and 2 are correct and that the mixture consists of two these compounds. The
retention times of 1 and 2 in the mixture separated by RP-HPLC are consistent with those of
synthetic dictyobispyrone B and E, respectively (see Supplementary Information). To confirm their
absolute stereochemistry at C-6, the specific optical rotation values of synthetic 1 and 2 and the
natural mixture were evaluated. The optical rotation values for 1 and 2 were [α]20 +38.7 (c 0.90,
D
CHCl3) and [α]20D +38.0 (c 1.16, CHCl3), respectively, while that of the natural mixture was [α]20
D
+40.7 (c 0.18, CHCl3). The agreement between their values suggests that the absolute
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stereochemistry at C-6 of 1 and 2 in the natural mixture is the same as that of synthetic
dictyobispyrone B and E.
Dictyobispyrone B (1) and E (2) possess an unprecedented α,α-bispyrone skeleton. Bispyrone-
type natural compounds are rather rare in nature, with published reports on only a few fused α,γ-
bispyrone compounds such as radicinin and its derivatives.18 Dictyobispyrones can be
biosynthesized from two distinct polyketide chains. As shown in Scheme 2, the first pyrone ring of
dictyobispyrone was constructed via ester condensation followed by Claisen condensation of two
polyketide chains. Subsequently, the second pyrone ring formed via enolation and ester
condensation, thereby completing the final structure of dictyobispyrone. Furthermore,
dictyobispyrones could be converted to corresponding dictyopyrones by hydration followed by
decarboxylation. Although dictyopyrones have been previously isolated from cellular slime molds,
their biosynthetic pathway is still unknown.14 Known biosyntheses from two polyketide chains such
as those of dictyobispyrone and dictyopyrone are rather rare, with only a few published studies on
them.19,20
O
O
O
polyketide
synthesis
pathway
O
SCoA
O
SCoA
O
SCoA
O
H
ester
condensation
Claisen
condensation
O
O
S
CoA
-
10
OH
10
10
O
O
O
O
CoAS
O
O
O
H
H
O
O
O
O
O
O
enolation and ester
condensation
OH
10
hydration
decarboxylation
10
10
OH
O
O
O
Dictyopyrone B
Dictyobispyrone B
Scheme 2. Plausible pathway for the biosyntheses of dictyobispyrone B and dictyopyrone B.
We conducted screening to investigate the biological activities of dictyobispyrones. We found
that dictyobispyrones at a concentration of 20 ꢀM inhibit histamine production in mouse
macrophage-like RAW 264.7 cells (Figure 4), although they do not exhibit antibacterial and
antiproliferative activities in mouse osteosarcoma LM8 and human epithelial carcinoma HeLa cells.
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Histamine is a mediator involved in inflammatory responses. It is produced by basophils and mast
cells in nearby infected tissues as part of the immune response to foreign pathogens. Hence,
dictyobispyrones may be a promising starting point for the development of antiallergic drugs.
Figure 4. Effects of dictyobispyrone B (A) and dictyobispyrone E (B) on lipopolysaccharide (LPS)-
induced histamine production in RAW 264.7 cells.
Cells were pre-incubated for 20 min with 0.1% DMSO (Control), 1 ꢀM, 5 ꢀM or 20 ꢀM of each dictyobispyrone B and
E. After addition of LPS (final concentration: 10 ꢀg/ml), cells were further incubated for 16 hours and assayed for
histamine production. Results are presented as mean values (± S.D.) of triplicate determination (n = 3). *, P < 0.05; **,
P < 0.01 versus Control (by Welch's t-test).
3. Conclusion
In summary, Zn2+ induces the production of two new metabolites, dictyobispyrone B (1) and E
(2), in D. giganteum. Dictyobispyrones are the first natural α,α-bispyrone-type compounds that
have been discovered. We propose their novel biosynthesis via cyclization of two distinct
polyketide chains. Our results suggest that cultivation of cellular slime molds in the presence of
Zn2+ is a useful technique for discovering more compounds that are structurally unique. Further
screening using Zn2+ will be carried out in the future to obtain other new compounds from other
species of cellular slime molds.
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4. Experimental section
General methods
Analytical TLC was performed on silica gel 60 F254 (Merck). Column chromatography was
carried out on silica gel 60 (70–230 mesh, Merck). NMR spectra were recorded on JEOL JNM
1
13
ECA-600 and AL-400. Chemical shifts for H and C NMR were given in parts per million (δ)
relative to tetramethylsilane (δH 0.00) and residual solvent signals (δC 77.0) as internal standards.
Mass spectra were measured on JEOL JMS-700, JMS-T100GC and JMS-DX303. HPLC was
carried on LC-908 (Japan Analytical Industry Co. Ltd.), using the column YMC-GPC-T2000 (φ 20
mm x 600 mm) (YMC Co. Ltd.). RP-HPLC was carried on Hitachi LaChrom system, column
Mightysil RP-18 GP 250-4.6 (5 ꢀm) (Kanto Chemical Co. Inc.). Optical rotations were measured
on JASCO P-1030 polarimeter. IR spectra were recorded on JASCO-FT/IR-4200 spectrometer.
Organism and culture conditions
The cellular slime molds, D. giganteum were kindly supplied by Dr. Hagiwara, National Science
Museum, Tokyo, Japan. The strain has been deposited into NBRP Nenkin (http://nenkin.nbrp.jp/)
(NBRP ID S90544). Spores were cultured at 22 °C with Klebsiella sp. on A-medium consisting of
0.5% glucose, 0.5% polypeptone, 0.05% yeast extract, 0.225% KH2PO4, 0.05% Na2HPO4, 0.05%
MgSO4·7H2O, 1.5% agar with (or without) several concentrations of ZnCl2. When fruiting bodies
had formed after 4 days, they were harvested for extraction.
Profiling metabolite production by RP-HPLC
Fruiting bodies were extracted three times with methanol at room temperature to give methanol
extract, which was then partitioned between ethyl acetate and water to yield ethyl acetate soluble.
Metabolites in ethyl acetate soluble were profiled using a RP-HPLC Hitachi LaChrom system,
column Mightysil RP-18 GP 250-4.6 (5 ꢀm) (Kanto Chemical Co., Inc.); flow : 1 mL/min; elutant :
acetonitrile-water (20:80) to acetonitrile-water (100:0) in 35 minutes; detection : UV 210 nm.
Samples were prepared in methanol solution and optimized amounts were injected to RP-HPLC
system for analysis.
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Isolation of dictyobispyrone B (1) and E (2)
The fruiting bodies of D. giganteum collected after cultured in A-medium with 0.5 mM ZnCl2
(dry weight 43.74 g) were extracted three times with methanol at room temperature to give
methanol extract (5.7 g). Methanol extract was then partitioned between ethyl acetate and water to
yield ethyl acetate soluble (686.4 mg). The ethyl acetate part was chromatographed over silica gel,
and the column was eluted with hexane–ethyl acetate and ethyl acetate-methanol with increasing
polarity to afford hexane–ethyl acetate (1:1) eluent (fraction A, 49.4 mg) and hexane–ethyl acetate
(3:1) eluent (fraction B, 196.1 mg).
Fraction A was subjected to recycle preparative HPLC (column : T2000, elutant : ethyl acetate,
detection : UV 254 nm, flow : 8 mL/min) to give fraction C. Fraction C was purified again by ODS
column eluted by water-acetonitrile (3:7) to give mixture of dictyobispyrone B (1) and E (2) (1.5
mg) (1 : 2 = 2:1) (Ratio was calculated on the basis of integrated values in 1H NMR spectrum and
1
peak area in RP-HPLC chart). Colorless amorphous solid; [α]25 +40.7 (c 0.18, CHCl3); H and
D
13C NMR data of the mixture, see Table S1 (Supplementary Information) (Although 1 and 2 could
be separated by RP-HPLC, the amount of individual compound was not enough for spectroscopic
analysis except mass spectrometry experiments); HREIMS m/z 360.2293 [M]+ (360.2299 calcd for
C22H32O4) and m/z 348.2293 [M]+ (348.2299 calcd for C21H32O4); FABMS m/z 361.3 [M+H]+
(361.2377 calcd for C22H33O4) and m/z 349.3 [M+H]+ (349.2377 calcd for C21H33O4).
Isolation of dictyopyrone B (3) and E (4)
Fraction B was subjected to recycle preparative HPLC (column : T2000, elutant : ethyl acetate,
detection : UV 254 nm, flow : 8 mL/min) to give fraction D, which was purified again two times by
silica gel column eluted by hexane–ethyl acetate (9:1) and ODS column eluted by water-acetonitrile
(3:7) to give mixture of dictyopyrone B (3) and E (4) (2.4 mg) (3 : 4 = 2:1). (Ratio was calculated
1
on the basis of integrated values in H NMR spectrum and peak area in HPLC chart). Colorless
amorphous solid; [α]25 +51.3 (c 0.26, CHCl3); 1H and 13C NMR data, see Table S2
D
(Supplementary Information) (Although 3 and 4 could be separated by RP-HPLC, the amount of
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individual compound was not enough for spectroscopic analysis except mass spectrometry
experiments); HREIMS m/z 334.2490 [M]+ (334.2506 calcd for C21H34O3) and m/z 322.2505 [M]+
(322.2506 calcd for C20H34O3); FABMS m/z 335.3 [M+H]+ (335.2584 calcd for C21H35O3) and m/z
323.3 [M+H]+ (323.2584 calcd for C20H35O3).
Synthesis of ethyl (S)-5-(methoxymethoxy)hex-2-ynoate (7)
n-Butyllithium in hexane (1.6 M) (1800 µL, 2.88 mmol) was added to a solution of ethyl
propiolate (292 µL, 2.88 mmol) in boron trifluoride ethyl etherate (800 µL, 1.44 mmol) at -78 ˚C.
After being stirred for 1 hour, (S)-(-)propylene oxide (100 µL, 1.44 mmol) was added to the mixture.
After being stirred for 3 hours, the mixture was poured into saturated ammonium chloride solution
and was extracted with dichloromethane three times. The organic layer was washed with brine,
dried over anhydrous sodium sulfate and evaporated. The residue was chromatographed over silica
gel eluted by hexane-ethyl acetate (4:1) to give the fraction (110.6 mg). To solution of this fraction
in dichloromethane (5 mL), chlorodimethyl ether (77 µL, 1.03 mmol) and N,N-
diisopropylethylamine (265 µL, 1.54 mmol) were added. After being stirred for 16 hours, the
mixture was evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl
acetate (9:1) to give ethyl (S)-5-(methoxymethoxy)hex-2-ynoate (7) (112.4 mg) (yield 39%, 2 steps).
1
Colorless oil; IR νmax (KBr) 2236, 1712, 1254, 1035 cm-1; H NMR (400 MHz, CDCl3) δ 4.68
(1H, d, J = 12.8 Hz), 4.66 (1H, d, J = 12.8 Hz), 4.21 (2H, q, J = 7.2 Hz), 3.92 (1H, m), 3.39 (3H, s),
2.60 (1H, dd, J = 5.6, 16.8 Hz), 2.52 (1H, dd, J = 6.4, 16.8 Hz), 1.31 (3H, d, J = 6.4 Hz), 1.30 (3H, t,
13
J = 7.2 Hz); C NMR (100 MHz, CDCl3) δ 153.6, 95.1, 85.7, 74.6, 70.8, 61.8, 55.4, 26.9, 20.1,
14.0; EIMS: m/z 156, 141, 128, 111, 89 (base); HRFABMS : m/z 201.1128 [M+H]+ (201.1126
calcd for C10H17O4).
Synthesis of methyl (E)-3-oxohexadec-14-enoate (8)
Meldrum's acid (26.7 mg, 0.19 mmol), N,N-dimethyl-4-aminopyridine (34.0 mg, 0.28 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (53.3 mg, 0.28 mmol) were added
to a solution of known compound (E)-tetradec-12-enoic acid (S1)16 (292 µL, 2.88 mmol) in
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dichloromethane (1.5 mL) at room temperature. After being stirred for 12 hours, 1 M hydrochloric
acid solution (1 mL) was added. The mixture was poured into water (50 mL) and extracted with
ethyl acetate (50 mL) three times. The organic layer was washed with brine, dried over anhydrous
sodium sulfate and evaporated. After methanol (10 mL) was added to the residue, the solution was
refluxed for 3 hours and evaporated. The residue was chromatographed over silica gel eluted by
hexane–ethyl acetate (19:1) to give methyl (E)-3-oxohexadec-14-enoate (8) (37.8 mg) (yield 72%).
1
Colorless amorphous solid; IR νmax (KBr) 2920, 2849, 1747, 1711, 1470 cm-1; H NMR (400
MHz, CDCl3) δ 5.39-5.42 (2H, m), 3.74 (3H, s), 3.45 (2H, s), 2.53 (2H, t, J = 7.5 Hz), 1.92-1.99
13
(2H, m), 1.63 (3H, d, J = 7.0 Hz), 1.54-1.62 (2H, m), 1.21-1.37 (14H, m); C NMR (100 MHz,
CDCl3) δ 202.8, 167.7, 131.6, 124.5, 52.3, 49.0, 43.0, 32.6, 29.6, 29.5, 29.4, 29.4, 29.3, 29.1, 28.9,
23.4, 17.9; EIMS: m/z 282 [M]+, 190, 129, 116 (base); HREIMS: m/z 282.2180 [M]+ (282.2193
calcd for C17H30O3).
Synthesis of methyl (S,E)-4-(2-(methoxymethoxy)propyl)-2-oxo-6-(tridec-11-en-1-yl)-2H-
pyran-5-carboxylate (9)
Compound 7 (26.0 mg, 0.13 mmol) and sodium methoxide (2.7 mg, 0.05 mmol) were added to
compound 8 (37.0 mg, 0.13 mmol) at room temperature. After being stirred for 3 hours at 100 ˚C,
acetyl acetate (0.5 mL) was added at room temperature. The solution was filtered through celite and
evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl acetate (9:1)
to give methyl (S,E)-4-(2-(methoxymethoxy)propyl)-2-oxo-6-(tridec-11-en-1-yl)-2H-pyran-5-
carboxylate (9) (44.6 mg) (yield 79%).
1
Colorless amorphous solid; IR νmax (KBr) 2928, 2854, 1731, 1033 cm-1; H NMR (400 MHz,
CDCl3) δ 6.08 (1H, s), 5.39-5.43 (2H, m), 4.61 (1H, d, J = 6.8 Hz), 4.49 (1H, d, J = 6.8 Hz), 3.78-
3.88 (1H, m), 3.86 (3H, s), 3.27 (3H, s), 2.82 (1H, dd, J = 8.0, 13.6 Hz), 2.64 (1H, dd, J = 7.5, 13.6
Hz), 2.58-2.66 (2H, m), 1.92-1.99 (2H, m), 1.65-1.72 (2H, m), 1.64 (3H, d, J = 7.0 Hz), 1.22-1.34
13
(14H, m), 1.20 (3H, d, J = 6.0 Hz); C NMR (100 MHz, CDCl3) δ 167.6, 166.2, 160.8, 154.6,
131.6, 124.5, 113.0, 112.8, 94.7, 71.7, 55.3, 52.3, 41.1, 32.9, 32.5, 29.5, 29.5, 29.4, 29.4, 29.2, 29.1,
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29.1, 27.6, 20.4, 17.9; FABMS: m/z 437.3 [M+H]+; HRFABMS: m/z 437.2903 [M+H]+ (437.2901
calcd for C25H41O6).
Synthesis of dictyobispyrone B (1)
p-Toluensulfonic acid (6.1 mg, 0.032 mmol) was added to a solution of compound 9 (35.0 mg,
0.080 mmol) in methanol (2 mL). After being stirred for 6 hours at 50 ˚C, the solution was
evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl acetate (4:1)
to give dictyobispyrone B (1) (20.0 mg) (yield 69%).
Colorless amorphous solid; [α]25 +38.7 (c 0.90, CHCl3); IR νmax (KBr) 2924, 2853, 1755, 1719,
D
1631, 1552, 1260, 1068 cm-1; 1H NMR (400 MHz, CDCl3) δ 5.99 (1H, s), 5.39-5.42 (2H, m), 4.47-
4.53 (1H, m), 3.03-3.14 (2H, m), 2.78 (1H, dd, J = 2.4, 16.2 Hz), 2.68 (1H, ddd, J = 1.8, 10.2, 16.2
Hz), 1.92-1.97 (2H, m), 1.65-1.79 (2H, m), 1.62-1.64 (3H, m), 1.47 (3H, d, J = 6.3 Hz), 1.35-1.41
13
(2H, m), 1.21-1.33 (12H, m); C NMR (100 MHz, CDCl3) δ 177.9, 162.1, 159.6, 153.2, 131.7,
124.5, 108.1, 104.8, 72.8, 35.8, 33.2, 32.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.2, 29.1, 27.5, 20.6, 17.9;
EIMS: m/z 360 [M]+, 315, 207 (base), 194; HREIMS: m/z 360.2287 [M]+ (360.2299 calcd for
C22H32O4).
Synthesis of methyl 3-oxopentadecanoate (10)
Meldrum's acid (288.3 mg, 2.0 mmol), N,N-dimethyl-4-aminopyridine (390.9 mg, 3.2 mmol) and
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (575.1 mg, 3.0 mmol) were added
to a solution of tridecanoic acid (428.7 mg, 2.0 mmol) in dichloromethane (10 mL) at room
temperature. After being stirred for 18 hours, 1 M hydrochloric acid solution (4 mL) was added.
The mixture was poured into water (100 mL) and was extracted with ethyl acetate (50 mL) three
times. The organic layer was washed with brine, dried over anhydrous sodium sulfate and
evaporated. After methanol (10 mL) was added to the residue, the solution was refluxed for 3 hours
and evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl acetate
(19:1) to give methyl 3-oxopentadecanoate (10) (270.2 mg) (yield 50%).
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1
Colorless amorphous solid; IR νmax (KBr) 2919, 2849, 1749, 1708 cm-1; H NMR (400 MHz,
CDCl3) δ 3.74 (3H, s), 3.45 (2H, s), 2.53 (2H, t, J = 7.2 Hz), 1.59 (2H, m), 1.22-1.32 (18H, m), 0.88
(3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 202.8, 167.6, 52.3, 49.0, 43.0, 31.9, 29.6, 29.6,
29.5, 29.4, 29.3, 29.1, 29.0, 23.4, 22.6, 14.1; EIMS: m/z 270 [M]+, 197, 129, 116 (base); HREIMS:
m/z 270.2184 [M]+ (270.2193 calcd for C16H30O3).
Synthesis of methyl (S)-6-dodecyl-4-(2-(methoxymethoxy)propyl)-2-oxo-2H-pyran-5-
carboxylate (11)
Compound 7 (60.0 mg, 0.3 mmol) and sodium methoxide (5.4 mg, 0.1 mmol) were added to
compound 10 (82.0 mg, 0.3 mmol) at room temperature. After being stirred for 3 hours at 100 ˚C,
acetyl acetate (0.5 mL) was added at room temperature. The solution was filtered through celite and
evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl acetate (9:1)
to give methyl (S)-6-dodecyl-4-(2-(methoxymethoxy)propyl)-2-oxo-2H-pyran-5-carboxylate (11)
(73.7. mg) (yield 58%).
1
Colorless oil; IR νmax (KBr) 2925, 2854, 1731, 1541, 1076, 1034 cm-1; H NMR (400 MHz,
CDCl3) δ 6.08 (1H, s), 4.61 (1H, d, J = 7.2 Hz), 4.49 (1H, d, J = 7.2 Hz), 3.86 (3H, s), 3.79-3.89
(1H, m), 3.27 (3H, s), 2.82 (1H, dd, J = 7.6, 13.6 Hz), 2.60 (1H, dd, J = 7.6, 13.6 Hz), 2.58-2.66
(2H, m), 1.65-1.73 (2H, m), 1.22-1.35 (18H, m), 1.20 (3H, d, J = 7.2 Hz), 0.88 (3H, t, J = 6.4 Hz);
13C NMR (100 MHz, CDCl3) δ 167.6, 166.2, 160.9, 154.7, 113.0, 112.8, 94.7, 71.7, 55.4, 52.4, 41.1,
33.0, 31.9, 29.6, 29.6, 29.6, 29.4, 29.3, 29.2, 29.2, 27.7, 22.7, 20.5, 14.1; FABMS: m/z 425.3
[M+H]+; HRFABMS: m/z 425.2903 [M+H]+ (425.2901 calcd for C24H41O6).
Synthesis of dictyobispyrone E (2)
p-Toluensulfonic acid (4.0 mg, 0.021 mmol) was added to a solution of compound 11 (44.3 mg,
0.104 mmol) in methanol (2 mL). After being stirred for 6 hours at 50 ˚C, the solution was
evaporated. The residue was chromatographed over silica gel eluted by hexane–ethyl acetate (4:1)
to give dictyobispyrone E (1) (33.1 mg) (yield 91%).
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Colorless amorphous solid; [α]25 +38.0 (c 1.16, CHCl3); IR νmax (KBr) 2918, 2843, 1748, 1731,
D
1
1712, 1551 cm-1; H NMR (400 MHz, CDCl3) δ 5.98 (1H, s), 4.48-4.54 (1H, m), 3.00-3.14 (2H, m),
2.79 (1H, dd, J = 2.9, 16.5 Hz), 2.69 (1H, ddd, J = 1.7, 10.7, 16.3 Hz), 1.63-1.80 (2H, m), 1.46 (3H,
13
d, J = 6.3 Hz), 1.33-1.41 (2H, m), 1.19-1.28 (16H, m), 14.1 (3H, d, J = 6.7 Hz); C NMR (100
MHz, CDCl3) δ 177.8, 162.0, 159.6, 153.2, 108.1, 104.8, 72.8, 35.7, 33.1, 31.9, 29.6, 29.6, 29.5,
29.4, 29.4, 29.3, 29.2, 27.5, 22.6, 20.6, 14.1; EIMS: m/z 348 [M]+, 315, 207 (base), 194; HREIMS:
m/z 348.2286 [M]+ (348.2299 calcd for C21H32O4).
Assay for histamine production by RAW 264.7 cells
Mouse macrophage-like RAW264.7 cells were pre-incubated for 20 min with DMSO (vehicle) or
the additives in 96-well tissue culture plate, each well containing 100 ꢀl of MEM medium (Sigma,
St. Louis, MO). After addition of LPS, cells were further incubated for 16 hours. Fifty microliter
each of the supernatant was used for the assay for histamine concentration by the use of Histamine
Test System (Kikkoman Biochemifa Company, Tokyo, Japan).
Acknowledgements
This work was supported in part by the Grants-in-Aid for Scientific Research (no. 16H03279)
from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan; the
Platform Project for Supporting in Drug Discovery and Life Science Research from Japan Agency
for Medical Research and Development (AMED); the Shorai Foundation for Science and
Technology; Kobayashi International Scholarship Foundation; and the Takeda Science Foundation.
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