Isolation and Structure Determination of New Pyrones from Dictyostelium spp. Cellular Slime Molds Coincubated with Pseudomonas spp.

Cellular slime molds are excellent model organisms in the field of cell and developmental biology because of their simple developmental patterns. During our studies on the identification of bioactive molecules from secondary metabolites of cellular slime molds toward the development of novel pharmaceuticals, we revealed the structural diversity of secondary metabolites. Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without using medium components. Although changing the feeding bacteria is expected to affect dramatically the secondary metabolite production, the effect of the feeding bacteria on the production of secondary metabolites is not known. Herein, we report the isolation and structure elucidation of clavapyrone (1) from Dictyostelium clavatum, intermedipyrone (2) from D. magnum, and magnumiol (3) from D. intermedium. These compounds are not obtained from usual cultural conditions with Klebsiella aerogenes but obtained from coincubated conditions with Pseudomonas spp. The results demonstrate the diversity of the secondary metabolites of cellular slime molds and suggest that widening the range of feeding bacteria for cellular slime molds would increase their application potential in drug discovery.


Introduction
Cellular slime molds are soil microorganisms that belong to the eukaryotic kingdom Amoebozoa, which is taxonomically distinct from fungi [1,2].As one of the most famous cellular slime molds owing to its simple developmental patterns and ease of handling, Dictyostelium discoideum has been used as a model organism for studying cell and developmental biology.In addition, considering cellular slime molds as a source of natural compounds, we isolated natural compounds with unique structures and biological activities [3][4][5][6][7][8][9].For example, brefelamide inhibits osteopontin expression [5], and ppc-1 is a promising candidate for antiobesity drugs [9].In 2005, the genome D. discoideum was found to contain approximately 45 polyketide synthase (PKS) gene clusters [1], and another cellular slime mold species, D. purpureum, was predicted to have 50 PKS genes [10].The number of genes in these organisms is higher than that observed in Streptomyces avermitilis, which is known to contain abundant secondary metabolites.Thus, we focused on cellular slime molds as a source of natural compounds to identify bioactive compounds in these organisms for the development of new drugs, revealing the structural diversity of their secondary metabolites.However, despite their usefulness, cellular slime molds have been underutilized in drug discovery compared with fungi, actinomycetes, and proteobacteria.
The production of secondary metabolites is highly dependent on the fermentation conditions, which provide the nutrients necessary for growth, such as carbon, nitrogen, and phosphate.Numerous metals such as zinc, iron, and manganese are also essential for bacterial growth, some of which also affect antibiotic production [11].Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without requiring medium components, whereas little is known about their growth by feeding on other bacteria [12,13].In fact, the effect of feeding bacteria on the production of secondary metabolites is unknown.Recently, our group isolated monochasiol F and G from D. monochasioides fed with K. aerogenes [3].These resorcinols contain unusual alkyl chains with a cyclopropane moiety.The origin of this substructure is considered to be lactobacillic acid, which is widely present in some bacteria.This result shows that coincubating bacteria directly affects the second metabolites of cellular slime molds [3].Here, we describe the structural elucidation of clavapyrone (1), intermedipyrone (2), and magnumiol (3), which were newly isolated from D. magnum, D. clavatum, and D. intermedium, respectively, and cultured with bacteria of the genus Pseudomonas instead of the one that is normally used (Figure 1).avermitilis, which is known to contain abundant secondary metabolites.Thus, we focused on cellular slime molds as a source of natural compounds to identify bioactive compounds in these organisms for the development of new drugs, revealing the structural diversity of their secondary metabolites.However, despite their usefulness, cellular slime molds have been underutilized in drug discovery compared with fungi, actinomycetes, and proteobacteria.
The production of secondary metabolites is highly dependent on the fermentation conditions, which provide the nutrients necessary for growth, such as carbon, nitrogen, and phosphate.Numerous metals such as zinc, iron, and manganese are also essential for bacterial growth, some of which also affect antibiotic production [11].Cellular slime molds grow by feeding on bacteria, such as Klebsiella aerogenes and Escherichia coli, without requiring medium components, whereas little is known about their growth by feeding on other bacteria [12,13].In fact, the effect of feeding bacteria on the production of secondary metabolites is unknown.Recently, our group isolated monochasiol F and G from D. monochasioides fed with K. aerogenes [3].These resorcinols contain unusual alkyl chains with a cyclopropane moiety.The origin of this substructure is considered to be lactobacillic acid, which is widely present in some bacteria.This result shows that coincubating bacteria directly affects the second metabolites of cellular slime molds [3].Here, we describe the structural elucidation of clavapyrone (1), intermedipyrone (2), and magnumiol (3), which were newly isolated from D. magnum, D. clavatum, and D. intermedium, respectively, and cultured with bacteria of the genus Pseudomonas instead of the one that is normally used (Figure 1).
D. discoideum, DiPKS1, also referred to as Steely1, catalyzes the formation of 2-alkyl pyrone [15,16].This type I FAS-type III PKS fusion enzyme is common among most cel lular slime molds and is presumed to be involved in the synthesis of differentiation-in ducing factors [17,18].This kind of enzyme constructs the α-pyrone skeleton, which then undergoes O-geranylation to afford clavapyrone.Although 4-methoxy-6-alkylpyrone have been reported [19], clavapyrone is the first example of a naturally synthesized 4-O geranylated α-pyranoid.D. discoideum, DiPKS1, also referred to as Steely1, catalyzes the formation of 2-alkylpyrone [15,16].This type I FAS-type III PKS fusion enzyme is common among most cellular slime molds and is presumed to be involved in the synthesis of differentiationinducing factors [17,18].This kind of enzyme constructs the α-pyrone skeleton, which then undergoes O-geranylation to afford clavapyrone.Although 4-methoxy-6-alkylpyrones have been reported [19], clavapyrone is the first example of a naturally synthesized 4-Ogeranylated α-pyranoid.
The determination of the absolute configuration of 2 was accomplished by comparing the electronic circular dichroism (ECD) spectra and specific optical rotations of asymmetrically synthesized 3-alkyl-3,4-dihydroxy-8-hydroxyisocoumarins, which indicated that the carbon number of 3-alkyl chains in dihydroisocoumarins does not affect the sign of rotation [20][21][22].The ECD spectrum of 2 showed a negative Cotton effect at 258 nm, and the C-3 position of 2 was determined to have an R configuration (Figure 1 and Table S1) This determination was confirmed by comparing the experimental and calculated ECD spectrum (Figure S11).The determination of the absolute configuration of 2 was accomplished by comparing the electronic circular dichroism (ECD) spectra and specific optical rotations of asymmetrically synthesized 3-alkyl-3,4-dihydroxy-8-hydroxyisocoumarins, which indicated that the carbon number of 3-alkyl chains in dihydroisocoumarins does not affect the sign of rotation [20][21][22].The ECD spectrum of 2 showed a negative Cotton effect at 258 nm, and the C-3 position of 2 was determined to have an R configuration (Figure 1 and Table S1).This determination was confirmed by comparing the experimental and calculated ECD spectrum (Figure S11).

Biological Activities of Compounds 1 and 2
Next, we investigated the biological activities of the isolated compounds 1 and 2. Compound 1 exhibited moderate antiproliferative activity against human leukemia K562 cells (IC 50 17 µM, Figure 5), whereas compound 2 did not inhibit K562 cells, human cervical cancer HeLa cells, and mouse 3T3-L1 fibroblast cells (a model nontransformed cell line) (IC 50 > 20 µM).Compounds 1 and 2 at concentrations of up to 100 µM did not show apparent antibacterial activities for Gram-positive (Staphylococcus aureus) and Gramnegative (E.coli) bacteria.Studies on the biological activities of 3 are currently underway.

Biological Activities of Compounds 1 and 2
Next, we investigated the biological activities of the isolated compou Compound 1 exhibited moderate antiproliferative activity against human le cells (IC50 17 µM, Figure 5), whereas compound 2 did not inhibit K562 cells, cal cancer HeLa cells, and mouse 3T3-L1 fibroblast cells (a model nontransfor (IC50 > 20 µM).Compounds 1 and 2 at concentrations of up to 100 µM did parent antibacterial activities for Gram-positive (Staphylococcus aureus) and tive (E.coli) bacteria.Studies on the biological activities of 3 are currently un

Discussion
In this study, we isolated three novel compounds from the fruiting bodies of cellular smile molds cultured with Pseudomonas spp.and determined their structures.6-Alkylpyrone moiety in clavapyrone and 6-methylsalicylic acid in intermedipyrone are predicted to be biosynthesized by type-I iterative polyketide synthases (iPKS), which are typical of fungi [25,26].Several cellular slime molds of the genus Dictyostelium are known to possess abundant polyketide synthase gene clusters [1,15], which are likely activated via coincubation with Pseudomonas spp. to biosynthesize the compounds in the present study.Compound 1 possesses a geranylated pyrone structure, which has not been reported to date, and compound 3 exhibits a salicylate with a characteristic branched alkyl chain.Thus, this study demonstrates that cellular slime molds are a promising source of natural products and presents coculture with bacteria as a new method for obtaining novel compounds.Furthermore, since bacteria other than Pseudomonas spp.can be used to culture cellular slime molds [12,13], the number of species of cellular slime molds and feeding bacteria will be increased to demonstrate the usefulness of this organism for drug discovery.In addition, studies are currently underway using techniques, such as Molecular Networking [27], to objectively demonstrate differences in extracts due to changes in feeding bacteria, which will accelerate the exploration of novel compounds.

General Methods
Analytical TLC was performed on silica gel 60 F254 (Merck).Silica and octadecyl silica gel column chromatography was conducted using Biotage Sfär Silica High Capacity Duo (Biotage, Uppsala, Sweden) and Biotage Sfär C18 Duo eluted by Isolera (Biotage, Uppsala, Sweden).NMR spectra were recorded on a JEOL ECA-600 spectrometer and a Bruker AVANCE 600 spectrometer. 1H and 13 C NMR chemical shifts are given in parts per million (δ) relative to tetramethylsilane (δ H 0.00) or residual solvent signals (δ H 7.26, δ C 77.0) as internal standards.Mass spectra were measured using JEOL JMS-700 and JMS-DX303 spectrometers.ECD spectra were measured on a J-1100DS spectrometer.Optical rotations were measured using a JASCO P-1030 polarimeter.

Isolation of Clavapyrone (1)
The fruiting bodies (dry weight 62.6 g) of D. clavatum TNS-C-220 were collected after coincubation with P. fluorescens on an A-medium agar plate.They were extracted twice with methanol at room temperature to give an extract (15.5 g), which was then partitioned between ethyl acetate and water to yield an ethyl acetate-soluble fraction (2.94 g).The ethyl acetate-soluble fraction was chromatographed over silica gel by eluting with hexane-ethyl acetate mixtures with increasing polarity to afford fraction A using hexane-ethyl acetate (2:1) as the eluent.Fraction A was separated using an octadecyl silica gel column with a water-acetonitrile solvent system, and fraction B was obtained by eluting with water-acetonitrile (1:9).Fraction B was subjected to recycle preparative HPLC using a GPC-T-2000 column (φ 20 mm × 600 mm, YMC Co., Ltd., Kyoto, Japan) and ethyl acetate as the solvent to give clavapyrone (1; 8.4 mg).Data for 1 are as follows: colorless amorphous solid; 1 H NMR and 13 C NMR spectroscopic data are shown in Table 1; and HR-EIMS m/z 314.1868 [M] + (314.1881calculated for C 20 H 26 O 3 ).

Isolation of Intermedipyrone (2)
The fruiting bodies (dry weight 45.2 g) of D. intermedium S90506 were collected after coincubation with P. fluorescens on modified MGY-1 medium agar plates and then extracted three times with methanol at room temperature to give an extract (6.37 g).The extract was partitioned between ethyl acetate and water to yield an ethyl acetate-soluble fraction (2.14 g), which was chromatographed over silica gel using hexane-ethyl acetate mixtures with increasing polarity as the eluent to afford fraction C when eluting with hexane-ethyl acetate (19:1).Fraction C was separated using a silica gel column with a hexane-chloroform solvent system to give fraction D by eluting with hexane-chloroform (1:1).Fraction D was subjected to recycle preparative HPLC using a GPC-T-2000 column (φ 20 mm × 600 mm, YMC Co., Ltd.) and ethyl acetate as the solvent, affording intermedipyrone (2; 1.2 mg).Data for 2 are as follows: colorless amorphous solid; [α]24D −20.6 (c 0.15, chloroform); 1 H NMR and 13 C NMR spectroscopic data are shown in Table 2; and HR-EIMS m/z 332.2358 [M] + (332.2351calculated for C 21 H 32 O 3 ).

Isolation of Magnumiol (3)
The fruiting bodies (dry weight 41.9 g) of D. magnum C-113 were collected after coincubation with P. chororaphis on modified MGY-2 medium agar plates.Methanol extraction three times at room temperature gave an extract (5.55 g), which was partitioned between ethyl acetate and water to yield an ethyl acetate-soluble fraction (1.58 g).The ethyl acetatesoluble fraction was chromatographed over silica gel by eluting with hexane-ethyl acetate mixtures with increasing polarity to afford fraction E when eluting with hexane-ethyl acetate (2:1).Fraction E was subjected to octadecyl silica gel column chromatography using a water-acetonitrile solvent system to give fraction F with water-acetonitrile (0:1) as the eluent.Fraction F was subjected to preparative TLC with hexane-isopropanol (199:1) to give magnumiol (3; 0.6 mg).Data for 3 are as follows: colorless oil; 1 H NMR and 13 C NMR spectroscopic data are shown in Table 3; and HR-FABMS m/z 381.2380 [M + Na] + (381.2403calculated for C 23 H 34 O 3 Na).
Figure 2. Structure of 1 and representative 1 H-1 H COSY and HMBC correlations.
Figure 3. Structure of 2 and representative 1 H-1 H COSY and HMBC correlations.