Isolation and Biosynthesis of Preussin B, a Pyrrolidine Alkaloid from

Mar 4, 2014 - A new pyrrolidine alkaloid, preussin B (1), was isolated from the culture extract of the fungus Simplicillium lanosoniveum TAMA 173 alon...
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Isolation and Biosynthesis of Preussin B, a Pyrrolidine Alkaloid from Simplicillium lanosoniveum Takao Fukuda,† Yuri Sudoh,‡ Yuki Tsuchiya,‡ Toru Okuda,‡,§ and Yasuhiro Igarashi*,† †

Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan ‡ Mycology & Metabolic Diversity Research Center, Tamagawa University Research Institute, 6-1-1 Tamagawa Gakuen, Machida, Tokyo 194-8610, Japan § Hyphagenesis Inc., 6-2-37 Tamagawa Gakuen, Machida, Tokyo 194-0041, Japan S Supporting Information *

ABSTRACT: A new pyrrolidine alkaloid, preussin B (1), was isolated from the culture extract of the fungus Simplicillium lanosoniveum TAMA 173 along with the known congener preussin (2). The structure and absolute configuration of 1 were determined by spectroscopic analysis and spectral comparison with 2. Feeding experiments with 13C-labeled precursors revealed that the pyrrolidine ring of 1 was assembled from acetate and L-phenylalanine by a PKS− NRPS hybrid biosynthetic pathway.

F

ration experiments using 13C-labeled precursors. Herein, we report the structure determination of preussin B (1) and the results of labeling experiments.

ungal species that are mutualistic or parasitic to other organisms such as insects and plants are a rich source of biologically active natural products.1,2 A wide range of compounds have been found from the species in the families Cordycipitaceae and Clavicipitaceae such as Cordyceps and Claviceps,3 but there still exist genera not fully investigated in these clades. Simplicillium belonging to the family Cordycipitaceae is a fungicolous fungus.4 This species is found commonly associated with rusts, in particular the coffee rust, Hemileia vastatrix.5 The teleomorph of Simplicillium is linked to Torrubiella, known as an arthropod-pathogenic fungus.6 While several bioactive compounds are known from Torrubiella, there exists only one report on the secondary metabolites of Simplicillium. 7 As a part of our continuing chemical investigation on underexplored pathogenic fungi,8,9 preussin B (1), a new pyrrolidine alkaloid, was isolated from Simplicillium lanosoniveum TAMA 173 along with its known analogue preussin (2). Compound 1 exhibited weak antifungal activity against Saccharomyces cerevisiae with an IC50 value of 25 μg/mL. Compound 2 was originally isolated from Aspergillus ochraceus ATCC2294710 and Preussia sp.11 as an antifungal antibiotic and has been shown to have antiviral activity12 and induce apoptosis in human cancer cell lines by inhibiting cyclinE kinase.13 Owing to these pharmacological interests, 2 has been a target of total and analogue synthesis,14,15 while nothing is known about its biosynthesis. Pyrrolidine alkaloids are distributed from plants to microorganisms,16 but our knowledge on their biosynthesis is limited to a few examples.17,18 In this study, the biosynthetic origin of the 2,5-disubstituted pyrrolidine ring in preussins was explored through incorpo© 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The fungal strain was isolated from a corpse of an unidentified aphid collected at Ibaraki, Japan. On the basis of morphological characteristics and ITS sequence similarity, this fungus was identified as S. lanosoniveum. HPLC analysis of the 1-butanol extract of the whole culture broth showed two major metabolites, which were purified by silica gel and reversephase preparative HPLC, to yield 10 mg of preussin B (1) and 4 mg of preussin (2) from approximately 2 L of liquid culture. Compound 1 was obtained as a colorless, amorphous solid. The UV spectrum, with a strong absorption at 206 nm and a weak absorption around 250 nm, was indicative of the presence of a benzene ring.19 High-resolution ESITOFMS gave an [M + H]+ at m/z 290.2486 (Δ +0.8 mmu), corresponding to the molecular formula C19H31NO, which was corroborated by 1H and 13C NMR data (Table 1). The 13C NMR and DEPT spectral analysis of 1 in combination with HSQC data established 19 carbons attributable to six sp2 carbons (five are proton-bearing), three methines bearing nitrogen or oxygen, eight methylenes, one nitrogen-bonded methyl, and one aliphatic methyl group. Contiguous COSY correlations from H2-7 to H2-12 and the HMBC correlations from H3-19 (δH 2.34) to C-8 and C-11 established the N-methylpyrrolidine unit. The presence of a Received: October 29, 2013 Published: March 4, 2014 813

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Table 1. 1H and 13C NMR Data for Preussin B (1) in CDCl3 δCa

δH (J in Hz)b

1 2 3 4 5 6 7

14.1, CH3 22.7, CH2 31.8, CH2 29.3,d CH2 29.9,d CH2 26.3, CH2 34.9, CH2

8 9α 9β 10 11 12

65.8, CH 39.3, CH2

0.88, t (7.0) 1.20−1.37,e m 1.20−1.37,e m 1.20−1.37,e m 1.20−1.37,e m 1.20−1.37,e m 1.72, m 1.20−1.37,e m 2.13, m 2.20, ddd (13.5, 9.3, 6.5) 1.42, ddd (13.5, 6.5, 1.0) 3.81, brt (4.3) 2.29, ddd (10.0, 4.5, 4.3) 2.85,f dd (13.0, 4.5) 2.89,f dd (13.0, 10.0)

position

13 14, 18 15, 17 16 19

70.5, CH 73.6, CH 33.7, CH2 139.4, 129.4, 128.4, 126.1, 38.6,

qC CH CH CH CH3

7.29,f m 7.28,f m 7.20, m 2.34, s

of 1, which displayed the highest similarity to those for the natural (2S*,3S*,5R*)-isomer among the four possible synthetic isomers of preussin (2) (Table S1).20 In addition, both 1 and 2 showed positive optical rotation with mutually close absolute values (1: [α]26D +22 (c 0.19 in CHCl3); 2: [α]25D +29.3 (c 1.14 in CHCl3)20a). On the basis of these analytical results as well as the consideration on biosynthetic relationship, 1 and 2 were strongly suggested to have the same relative and absolute configurations. The structures of preussins are reminiscent of 3-acyltetramic acids (3-acyl-2,4-dioxopyrrolidine; Figure 2) represented by

HMBCb,c 2, 3

6

7, 8 7, 8, 10, 11 8 9 10, 11, 13, 14 (18) 10, 11, 13, 14 (18) 12 14 (18), 15 (17) 8, 11

a

Recorded at 100 MHz. bRecorded at 500 MHz. cHMBC correlations are from proton(s) stated to the indicated carbon. dChemical shifts are assigned using 13C-labeled 1 isolated from the feeding experiments with [1-13C]- and [2-13C]acetates. eOverlapping signals. f1H shifts are assigned by HSQC experiments.

monosubstituted benzene ring was confirmed by analysis of the 1 H, 13C, and HSQC spectra, showing relatively intense signals at δC 129.4 and 128.4, each accounting for two proton-bearing carbons (C-14/C-18 and C-15/C-17, respectively), and resonances at δH 7.20 and 7.28−7.29 accounting for five protons. This aromatic part was connected to C-12 on the basis of HMBC correlations from H2-12 to C-13 and C-14 (C-18). The remaining triplet methyl and five methylenes were assigned to the linear alkyl chain from C-1 to C-6, which was connected to C-7 by HMBC correlations from H2-7 to C-6 to complete the planar structure of 1 (Figure 1a).

Figure 2. Biosynthetic pathway for fungal tetramic acid and related metabolites.

militarinone C and epicoccarine A.21,22 These fungal tetramic acids are derived from a PKS−NRPS hybrid pathway,23,24 bearing an unsaturated aliphatic chain and a benzyl substitution at C-3 and C-5, respectively, of the 2,4-dioxopyrrolidine ring. In these compounds the tetramic acid moiety is assembled by the condensation of phenylalanine or tyrosine to the polyketide chain and a Claisen-type intramolecular cyclization associated with the release of an NRPS enzyme (Figure 2).25 Ring expansion from the tetramic acid by oxidative rearrangement provides a 2-pyridone ring system being substituted with an alkyl and an aromatic group as seen in tenellin.26 Additionally, tetramic acid is a key precursor for pseurotins and synerazol, unusual heterospirocyclic γ-lactams.27 Meanwhile, preussin (2) is the only fungal metabolite possessing a pyrrolidine ring in the center of the molecule instead of the aforementioned heterocyclic cores. 2 has a benzyl substitution at C-5 similar to tetramic acids, while a linear side chain is positioned at C-2 of the pyrrolidine ring. Despite this difference, the structural similarity of 2 to tetramic acids is indicative of the aromatic part including the nitrogen atom in the pyrrolidine ring being likely derived from phenylalanine and the remaining aliphatic part from the malonate pathway. In order to evaluate our hypothesis on the biogenesis of 1, feeding experiments with 13C-labeled compounds were carried out in a pulsed labeling manner. When [1-13C]acetate was

Figure 1. COSY, key HMBC, and NOESY correlations of preussin B (1).

The relative configuration of 1 was assigned by interpretation of the NOESY spectrum. NOEs were observed between H-9α and H-10, H-10 and H-11, and H-11 and H-8, indicating that the alkyl, hydroxy, and benzyl groups were placed on the same side of the pyrrolidine ring (Figure 1b). This assignment was supported by the proton chemical shifts for the pyrrolidine ring 814

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incorporated into 1, high levels of 13C enrichments were observed at C-2, C-4, C-6, and C-8. [2-13C]Acetate was incorporated into 1, labeling C-1, C-3, C-5, C-7, and C-9 with essentially equal enrichments. The remaining three carbons C10, C-11, and C-12 were not labeled by these precursors. When 13 13 L-[4- C]- and L-[1- C]phenylalanines were administered to the producing strain, significant enhancement of the 13C signal intensity was found at C-13 and C-10, respectively. These results established that the C-10−C-12 fragment and the benzene ring were derived from L-phenylalanine and the carbons from C-1 to C-9 from acetates (Table 2, Figure 3).

Hz, C-1: J = 34.4 Hz). Because the 2JCC values among the carbons C-1 to C-9 were mutually close, it was not possible to assign the intact acetate units only from the coupling constants. However, the 2D-INADEQUATE spectrum clearly gave crosspeaks for C-1/C-2, C-3/C-4, C-5/C-6, and C-7/C-8 (Figure S10), establishing the chain extension direction from C-1 to C9 with the deletion of one carbon from C-9 possibly by decarboxylation. On the basis of the results from labeling experiments, we propose two pathways for the pyrrolidine ring formation in preussin biosynthesis (Figure 4). The first one is illustrated as path A, in which a pentaketide and a phenylalanine are coupled to provide a ketoamine intermediate, which undergoes intramolecular cyclization by reductive amination. Another possible route to the pyrrolidine assembly is illustrated as path B. After the formation of a phenylalanine amide tetraketide, an additional malonate is conjugated and cyclization takes place through Dieckman-type condensation. To the best of our knowledge, neither pathway is known as a route to 2,5disubsitituted pyrrolidines. The monosubstituted pyrrolidine ring of L-proline is formed by intramolecular reductive amination of glutamate-γ-semialdehyde from L-glutamic acid.29 For the formation of a pyrrolidine ring in xenocoumacin II, a coumarine derivative from a bacterium, Xenorhabdus nematophila, intramolecular cyclization with the release of a guanidium group is proposed.17 The pyrrolidine ring of nicotine also arises from the cyclization of an aminoaldehyde precursor derived from L-ornitine.30 In contrast to these monosubstituted pyrrolidines, 2,5-disubstituted pyrrolidines are a small family of natural products,10,11,16 and very little is known about their biosynthesis. Brossonetine, a plant metabolite, is the only example of a 2,5-disubstituted pyrrolidine of which biosynthetic origins were studied. On the basis of results from a feeding experiment using 1-13C-D-glucose to the callus of Broussonetia kazinoki, it was proposed that acetates and serine are the carbon sources for the pyrrolidine moiety,31 although additional labeling experiments are necessary to prove this proposal.

Table 2. Incorporation of 13C-Labeled Precursors into 1 relative enrichmentsa

1 2 3 4 5 6 7 8 9 10 11 12 13 14/18 15/17 16 19

13

δC

[1- C] acetate

13

[2- C] acetate

13 L-[1- C] phenylalanine

13 L-[4- C] phenylalanine

14.1 22.6 31.8 29.3 29.8 26.4 34.2 66.2 39.3 70.2 74.0 33.2 139.1 128.4 129.4 126.2 38.5

1.00 3.58b 0.90 3.45 0.85 3.86 0.89 3.89 0.92 0.93 1.01 0.95 1.05 0.96 0.85 0.84 1.05

3.09 1.00 3.13 0.94 2.70 0.92 2.73 1.02 3.06 0.72 0.85 0.81 0.82 0.97 0.81 0.91 1.15

0.88 0.98 0.93 1.09 0.93 1.07 0.93 1.01 0.99 60.65 0.92 1.00 0.97 1.01 0.94 1.01 2.37

1.19 1.27 1.14 1.33 1.17 1.28 1.07 1.30 1.16 0.94 1.00 1.08 55.65 1.34 1.93 1.21 1.25

a13

C signal intensity of each peak in the labeled 1 divided by that of the corresponding signal in the unlabeled 1, respectively, normalized to give an enrichment ratio of 1 for the unenriched peak (C-1 for [1- 13 C]acetate, C-2 for [2- 13 C]acetate, C-12 for L -[1- 13 C]phenylalanine, and C-11 for L-[4-13C]phenylalanine). bThe numbers in bold type indicate 13C-enriched atoms from 13C-labeled precursors.

Figure 3. Incorporation of 13C-labeled precursors into 1.

The incorporation pattern of [1-13C]- and [2-13C]acetates suggested two possible directions of polyketide chain extension, C-1 to C-9 and C-9 to C-1, as well as the removal of one carbon of an acetate unit from either C-1 or C-9 associated with the skeletal formation. To discriminate these possibilities, it became necessary to assign the incorporation sites of the intact acetate units by labeling 1 with [1,2-13C2]acetate. The protondecoupled 13C NMR spectrum of 1 labeled with [1,2-13C2]acetate displayed complex splitting signals for C-2 to C-8 with the central singlet peaks due to natural acetates, doublet peaks due to the intact acetate unit, and the minor double of doublet signals due to the sequential incorporations of [1,2-13C2]acetate in the neighboring units (Figure S9).28 The resonances for C-1 and C-9 were observed as a singlet with satellites (C-9: J = 35.1

In summary, we have isolated preussin B (1), a new polyketide bearing a pyrrolidine ring from a fungus, and elucidated that the pyrrolidine moiety is assembled from a phenylalanine and malonates through incorporation experiments with 13C-labeled precursors.

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EXPERIMENTAL SECTION

General Experimental Procedures. Sodium [1-13C]- and [2-13C]acetates were purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA), and sodium [1,2-13C2]acetate and L[4-13C]phenlalanine from ISOTEC Inc. (Miamisburg, OH, USA). L[1-13C]Phenylalanine was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Optical rotations were measured using a 815

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Figure 4. Possible biosynthetic route to pyrrolidine ring formation in 1. dryness to give a TFA salt of preussin B (1, 10 mg, tR 29.8 min) and preussin (2, 4 mg, tR 32.5 min), respectivery. For the comparison of the optical rotation and 1H NMR data with reported values, salt-free 1 and 2 were obtained by washing their EtOAc extract with 0.1% NH4OH solution. Preussin B (1): colorless, amorphous solid; [α]26D +22 (c 0.19, CHCl3); UV (MeOH) λmax (log ε) 206 (4.04), 250 (2.39) nm; IR (ATR) νmax 3428, 2955, 2925, 2857 cm−1; for 1H and 13C NMR data, see Table 1; HRESITOFMS [M + H]+ 290.2486 (calcd for C19H32NO, 290.2478). Preussin (2): pale yellow, amorphous solid; [α]25D +20 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 206 (4.03), 250 (2.39) nm; IR (ATR) νmax 3428, 2955, 2925, 2857 cm−1; 1H and 13C NMR data were identical with those reported in the literature;20a HRESITOFMS [M + H]+ 318.2790 (calcd for C21H36NO, 318.2791). Feeding Experiments with 13C-Labeled Precursors. Feeding experiments were performed using L-[4-13C]- and L-[1-13C]phenylalanine and sodium [1-13C]-, [2-13C]-, and [1,2-13C2]acetate. In all cases, 13C-enriched carbon sources were dissolved in distilled water (12.5 mg/mL), and each solution was sterilized by filtration through a 0.2 μm filter (Minisart). A 200 μL aliquot was fed to 20 mL of growing cultures of S. lanosonibeum TAMA 173. Feeding of each labeled precursor (2.5 mg/flask) commenced two days after inoculation and continued three times at 48 h intervals. The fermentation was allowed to continue for 12 days, and the culture broth (20 mL × 48 flasks) was extracted as described above. Each extract was suspended in water (100 mL) and extracted with n-hexane (3 × 50 mL). Then, a 28% aqueous ammonia solution (100 μL) was added to the remaining water layer, which was further extracted with nhexane (3 × 50 mL). This hexane extract was concentrated and purified using RP-HPLC (CAPCELL PAK C8 DD S5 10 × 250 mm) with a gradient of MeCN−0.1% NH4OH (MeCN concentration: 65% for 0−3 min, 65−85% for 3−15 min, 85−100% for 15−25 min) to obtain 1 labeled with 13C-precursors (tR 16.2 min). 1. Sodium [1-13C]acetate. Sodium [1-13C]acetate (total 480 mg; 2.5 mg × 48 flasks × 4 days) was fed to 0.96 L of production medium, and 3.2 mg of 13C-labeled 1 was obtained. The 13C NMR spectrum showed enriched signals at δ 65.8, 29.3, 26.3, and 22.7. 2. Sodium [2-13C]acetate. Sodium [2-13C]acetate (total 480 mg; 2.5 mg × 48 flasks × 4 days) was fed to 0.96 L of production medium, and 6.0 mg of 13C-labeled 1 was obtained. The 13C NMR spectrum showed enriched signals at δ 39.3, 34.9, 31.8, 29.9, and 14.1. 3. Sodium [1,2-13C]acetate. Sodium [1,2-13C]acetate (total 480 mg; 2.5 mg × 48 flasks × 4 days) was fed to 0.96 L of production medium, and 6.7 mg of 13C-labeled 1 was obtained. 4. L-[1-13C]Phenylalanine. L-[1-13C]Phenylalanine (total 480 mg; 2.5 mg × 48 flasks × 4 days) was fed to 0.96 L of production medium, and 10.9 mg of 13C-labeled 1 was obtained. The 13C NMR spectrum showed an enriched signal at δ 70.5.

JASCO P-1030 polarimeter. UV spectra were recorded on a Hitachi U3210 spectrophotometer. IR spectra were measured on a Perkin-Elmer Spectrum 100. NMR spectra were obtained on a Bruker AVANCE 500 spectrometer or Bruker AVANCE 400 spectrometer. 1H and 13C chemical shifts were referenced to the solvent signals in CDCl3 (δH = 7.26, δC = 77.0). HRESITOFMS were measured on a Bruker microTOF spectrometer. HPLC analyses were carried out on an Agilent HP1100 system using a CAPCELL PAK C8 DD S5 (Shiseido Co., Ltd., 4.6 × 250 mm). Silica gel 60 (Kanto Chemical Co., Inc., 63210 μm) was used for silica gel column chromatography. HPLC separation was performed using a CAPCELL PAK C8 DD S5 (Shiseido Co., Ltd., 10 × 250 mm). Microorganism. The strain TAMA 173 was isolated through the following process. An unidentified dead insect belonging to the family Aphidoidea collected from a leaf of Cinnamomum sp. in Tsukuba, Japan (Dec 18, 2003), was inoculated onto Miura medium32 containing 0.05% chloramphenicol and 0.01% streptomycin. After incubation at room temperature for three weeks, a fungal strain growing on the corpse was directly isolated. The strain was identified as Simplicillium lanosoniveum (F. H. van Beyma) Zare & W. Gams based on the long almost solitary phialides from prostrate hyphae, bearing short-ellipsoidal and small conidia.33 This morphological conclusion was supported by the molecular phylogeny analysis of the ITS sequence (609 nucleotides; DDBJ accession number AB758126). The heuristic search generated one most parsimonious (MP) tree (tree length: 864.78, consistency index: 0.626, homoplasy index: 0.3736, and retention index: 0.7766). In the tree, the strain was grouped with Cephalosporium lanosoniveum (Simplicillium lanosoniveum) IMI 317442 and CBS 704.86 (bootstrap value: 65) in the Simplicillium clade. The Simplicillium clade was supported by a 100% bootstrap value. Fermentation and Isolation. Strain TAMA 173 grown on a slant culture was inoculated into 150 mL polypropylene flasks each containing 20 mL of the production medium consisting of sucrose 8%, yeast extract 0.1% (Difco Laboratories), and corn meal 0.5%. The inoculated flasks were placed on a rotary shaker (225 rpm) at 25 °C for 14 days. The whole culture broth of strain TAMA 173 (20 mL × 96 flasks) was extracted with 1-butanol (15 mL/flask) on a rotary shaker (225 rpm) for 1 h. The mixture was centrifuged, and the organic layer was separated from the aqueous layer containing the mycelium. Evaporation of the organic solvent provided approximately 3.5 g of crude extract, which was chromatographed on a silica gel column with CHCl3−MeOH (1:0, 20:1, 10:1, 4:1, 2:1, 1:1, and 0:1 v/ v). Fraction 4 (4:1) was evaporated to give a crude material, which was further purified by preparative HPLC (CAPCELL PAK C8 DD S5, 10 × 250 mm, 3.7 mL/min) with a gradient of MeCN−0.1% trifluoroacetic acid (MeCN concentration: 20% for 0−3 min; 20− 48% for 3−13 min; 48% for 13−24 min; 48−100% for 24−27 min; 100% for 27−45 min), and the collected fraction was concentrated to 816

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5. L-[4-13C]Phenylalanine. L-[4-13C]Phenylalanine (total 480 mg; 2.5 mg × 48 flasks × 4 days) was fed to 0.96 L of production medium, and 6.0 mg of 13C-labeled 1 was obtained. The 13C NMR spectrum showed an enriched signal at δ 139.5.

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ASSOCIATED CONTENT

S Supporting Information *

NMR data, 1D/2D NMR spectra; 13C NMR spectra of 1 derived from 13C-labeled precursors. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-766-56-7500. Fax: +81-766-56-2498. E-mail: yas@ pu-toyama.ac.jp. Notes

The authors declare no competing financial interest.

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