Biosynthesis of Resorcylic Acid Lactone Lasiodiplodin in Lasiodiplodia Theobromae

Biosci. Biotechnol. Biochem., 73 (5), 1118–1122, 2009

Biosynthesis of Resorcylic Acid Lactone Lasiodiplodin in Lasiodiplodia theobromae

y Takasumi KASHIMA, Kosaku TAKAHASHI, Hideyuki MATSUURA, and Kensuke NABETA

Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received December 10, 2008; Accepted January 26, 2009; Online Publication, May 7, 2009 [doi:10.1271/bbb.80878]

The biosynthesis of lasiodiplodin (1) and its (5S)-5- as an example, the initial PKS was of the reducing hydroxylated derivative (2) were investigated by the type (R-PKS) named PKS4, containing minimal PKS administration of 13C-labeled acetates to Lasiodiplodia condensation domains of ketosynthase (KS), malonyl- theobromae. The labeling patterns of biosynthetically CoA:ACP acyltransferase (MAT), and acyl carrier 13C-labeled 1 and 2 were determined by 13C-NMR and protein (ACP); additional domains include those of INADEQUATE spectra, demonstrating the octaketide dehydratase (DH), enoylreductase (ER), and ketoreduc- origins of 1 and 2. Taking into account the biosynthetic tase (KR). The subsequent PKS was nonreducing PKS study of resorcylic acid lactones, the involvement of (NR-PKS) named PKS13, containing a putative starter highly reduced acyl intermediates in the biosynthesis of unit-ACP transacylase (SAT), a putative product tem- lasiodiplodins was presumed; thus, we synthesized 2H- plate (PT), and C-terminal thioesterase (TE) domains in labeled hypothetical acyl intermediates of 1, 9-hydrox- addition to the minimal domains. The biosynthetic ydecanoic acid (4) and its N-acetylcysteamine thioester system for zearalenone starts with the reduced hexake- (SNAC, 5). When L. theobromae was incubated with tide acyl chain being synthesized by PKS4 and trans- 5mM of a 2H-labeled intermediate, the 2H-label from ferred to PKS13. PKS13 then extends the acyl chain the intermediate was incorporated at the expected with three acetyl-CoA units, and then facilitates an aldol position of 1. These incorporation studies revealed that condensation to yield the resorcylic acyl intermediate. 1 was produced via a pathway which closely resembles Finally, it undergoes macrocyclization, resulting in the that of resorcylic acid lactone biosynthesis. production of zearalenone. In the case of hypothemycin, the polyketide skeleton was presumably synthesized Key words: lasiodiplodin; biosynthesis; resorcylic acid along almost the same pathway as that for zearalenone. lactone; Lasiodiplodia theobromae The structures of lasiodiplodins are classified as those of a resorcylic acid lactone; however the 12-membered Resorcylic acid lactones (RALs) are members of a macrolactone ring is two carbons shorter than with the unique family of macrolides that possess such potent other RALs just described. Taking into consideration this biological activities as radicicol (Hsp90 inhibitor),1) structural similarity, lasiodiplodins are probably synthe- LL-Z1640-2 (TAK1 inhibitor),2) hypothemycin (MAP sized via the same biosynthetic system as other RALs. kinase inhibitor),3) and zearalenone (estrogen receptor We demonstrate in this report that lasiodiplodin (1) agonist).4) Despite their structural similarity, a highly and (5S)-5-hydroxylasiodiplodin (2) were synthesized reduced 14-membered macrolactone with resorcylate via a polyketide biosynthetic pathway in L. theobromae (2,4-dihydroxybenzoate) in its lactone ring, these RALs by the incorporation of 13C-labeled acetate. The incor- are mycotoxins produced by a variety of different fungal poration of the chemically synthesized 2H-labeled species via polyketide biosynthesis.5) precursors into 1 clearly indicates that lasiodiplodin Lasiodiplodin 1 (Fig. 1) was first reported as a was biosynthesized by a similar biosynthetic system to metabolite produced by the fungus, Lasiodiplodia such other RALs as zearalenone and hypothemycin. theobromae, functioning as a potent plant growth inhibitor,6) and potent antileukemic activity was later Results and Discussion characterized.7) Lasiodiplodia theobromae is a well- known fungus that produces the plant hormone, jas- Lasiodiplodia theobromae was statically incubated in monic acid,6) as well as other physiologically active a 1% potato-glucose medium for 7 d. Three kinds of 13 13 13 13 compounds such as theobroxide and its derivatives C-labeled sodium acetate ([1- C], [2- C], and [ C2]) which are active in flower bud formation and/or potato were separately supplemented to the culture at a micro-tuber formation,8–11) and a variety of lasiodiplodin concentration of 10 mM. After an additional 10-d derivatives active in potato micro-tuber formation.12–15) incubation, the culture was filtered to separate the The biosynthesis of zearalenone and hypothemycin mycelia and supernatant. The mycelia and supernatant has recently been genetically characterized, indicating were extracted with EtOAc. After those extracts had that these RALs were mainly synthesized by only two been combined and evaporated, the resulting residue polyketide synthases (PKSs).16–19) Taking zearalenone was separated twice by PTLC, affording pure biosyn-

y To whom correspondence should be addressed. Tel/Fax: +81-11-706-2505; E-mail: [email protected] Abbreviations: SNAC, N-acetylcysteamine thioester; RALs, resorcylic acid lactones; PKS, polyketide synthase; R-PKS, reducing PKS; KS, ketosynthase; MAT, malonyl-CoA:ACP acyltransferase; ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KR, ketoreductase; NR-PKS, nonreducing PKS Lasiodiplodin Biosynthesis in Lasiodiplodia theobromae 1119 13 13 13 Table 2. Incorporation of Sodium [1- C], [2- C], and [ C2] Acetate into 1

13C-atom% C Position C 13 13 13 (acetylated) [1- C] [2- C] [ C2] JC{C acetate acetate acetate (Hz) 1 169.5 167.8 3.70 0.97 3.77 77.1 3 72.6 72.3 3.43 1.12 3.37 39.1 4 32.3 32.3 0.90 4.78 3.63 35.2 5 21.3 21.2 3.81 1.12 3.59 35.2 6 26.3 26.4 0.88 5.58 3.53 35.2 7 24.1 24.2 3.84 1.17 3.80 34.6 Fig. 1. Structures of Lasiodiplodin 1 and (5S)-5-Hydroxylasiodiplo- 8 25.4 25.4 0.88 5.61 3.45 34.6 din 2. 9 30.0 29.9 3.53 1.21 3.61 34.6 10 30.4 30.4 0.87 4.95 3.48 42.5 10a 142.9 142.5 3.51 1.00 2.93 42.5 Table 1. Administration Experiments Using the 13C-Labeled Acetate 11 108.4 114.5 0.87 5.41 3.32 67.0 and 2H-Labeled Acyl Intermediate of Compounds 1 and 2 12 157.9 151.9 3.69 1.18 3.51 67.0 Yield 13 97.0 102.7 0.93 5.05 3.61 70.4 Amount Concentration 14 157.9 157.2 3.41 1.05 3.40 70.4 Precursor (mg/150 ml) (mg/150 ml) (mM) 14a 117.0 122.6 0.94 4.59 3.73 77.1 12 15 19.5 19.4 0.88 6.02 3.82 39.1 Sodium [1-13C] acetate 125 10 14.4 4.7 PhOMe 55.7 56.0 1.01 3.41 2.67 Sodium [2-13C] acetate 125 10 6.3 2.2 CH3CO–Ph 21.3 1.11 1.11 1.11 13 CH3C(=O)–Ph 169.0 Sodium [ C2] acetate 126 10 6.6 4.4 Compound 4 144 5 14.4 Compound 5 219 5 1.6 13 13 13 Table 3. Incorporation of Sodium [1- C], [2- C], and [ C2] Acetate into 2 thetically 13C-labeled 1 and 2. The yields of 1 and 2 were in the range of 6.3–14.4 mg and 2.2–4.7 mg, 13C-atom% C respectively, per 150 ml of culture (Table 1). Position C 13 13 13 (acetylated) [1- C] [2- C] [ C2] JC{C The 13C{1H}-NMR spectra of [1-13C] acetate-derived acetate acetate acetate (Hz) 1 and 2 showed several enhanced signals at C-1, C-3, 1 168.3 167.5 3.53 1.23 4.76 77.1 C-5, C-7, C-9, C-10a, C-12, and C-14. In contrast, those 3 69.6 69.4 3.78 1.23 5.18 39.1 13 of [2- C] acetate-derived 1 and 2 revealed enhanced 4 40.4 36.9 0.85 5.79 4.93 38.5 signals at C-4, C-6, C-8, C-10, C-11, C-13, C-14a, and 5 66.7 69.8 3.99 1.22 5.19 38.5 C-15. Labeled acetate-derived 1 and 2 were then 6 35.7 31.9 0.90 5.96 4.69 34.1 acetylated, and the 13C abundance ratio was calculated, 7 22.1 21.5 3.78 1.38 5.47 34.6 referencing the relative intensity of the acetyl methyl 8 24.9 24.8 0.86 5.99 4.64 34.6 9 30.1 29.8 3.77 1.59 4.73 34.1 signal of the C-12 hydroxyl group as 1.11% of natural 10 30.1 30.0 1.02 5.40 4.88 43.6 13 abundance. The specific incorporation of Cto1 and 2 10a 143.0 142.3 3.47 1.07 3.89 42.5 was in the range of 3.4–6.0% and 3.5–6.0%, respectively 11 108.4 114.6 0.84 5.65 4.65 67.0 (Tables 2 and 3). Moreover, the 13C{1H}-NMR spec- 12 157.5 152.1 3.83 1.25 4.75 67.0 13 13 97.0 102.8 0.83 5.38 4.66 70.4 trum of [ C2] acetate-derived 1 and 2 showed intense 13C–13C coupling signals besides a methoxyl carbon 14 158.2 157.3 3.82 1.24 4.47 70.4 14a 117.4 122.2 0.89 5.96 5.00 77.1 signal at C-14. The coupling constants of several 15 20.0 19.4 0.92 5.79 5.34 39.1 methylene carbons in the lactone ring were quite similar, PhOMe 55.9 56.0 0.99 3.74 3.26 13 13 making it difficult to recognize each pair of C– C CH3CO–Ph 21.3 1.11 1.11 1.11 signals. In order to obtain definitive evidence for the CH3C(=O)–Ph 169.0 1.06 1.07 1.13 13 13 C– C coupled pair, we measured INADEQUATE CH3CO–CH 21.1 1.07 1.20 1.26 13 CH C(=O)–CH 170.2 0.95 0.98 1.10 spectral data for [ C2] acetate-derived 1 and 2 (Fig. 2A 3 and B). Both INADEQUATE spectra demonstrated clear 13C–13C labeling patterns of eight intact acetate units, C-1/C-14a, C-14/C-13, C-12/C-11, C-10a/C-10, C-9/ Each of 2H-labeled precursors 4 and 5 dissolved in C-8, C-7/C-6, C-5/C-4, and C-3/C-15. These labeling DMSO was added to a 7-d-old culture of L. theobromae, patterns confirmed that the resorcylic acid lactone achieving a final concentration of 5 mM. After an skeletons of 1 and 2 were constructed from eight acetate additional 10-d incubation, compound 1 was isolated units via a polyketide biosynthetic pathway. by the same procedure as that for acetate-derived 1. The Since conclusive evidence for 1 and 2 being synthe- administration of 4 and 5 to 1 yielded 14.4 mg and sized via a polyketide biosynthetic pathway in L. theo- 1.6 mg/150 ml of culture, respectively. The yield of bromae had been obtained, we further demonstrated 1 from the culture fed with 5 was nine times lower whether lasiodiplodins were biosynthesized via highly than that without 5, while the administration of 4 did reduced acyl intermediates in the same manner as other not result in a lower yield of 1. The administration of 2 RALs. We synthesized [10,10,10- H3] 9-hydroxydeca- 5 also macroscopically retarded the cell growth of noic acid (4) and its N-acetylcysteamine thioester L. theobromae, suggesting that 5 might inhibit fatty acid (SNAC, 5) as indicated in Fig. 3. metabolism. 1120 T. KASHIMA et al. A

13 Fig. 2. INADEQUATE Spectra of [ C2] Acetate-Derived 1 (A) and 2 (B).

Fig. 3. Preparation of 4 and 5. a) O3,CH2Cl2, 78 C, 3 h. b) Me2S, r.t., overnight, 67% (2 steps). c) CD3MgI, Et2O, 0 C, overnight, 60%. d) DPPA, Et3N, DMF, 0 C, 2 h. e) HSNAC, r.t., overnight, 69% (2 steps).

The 2H-NMR spectral data for both resulting 4 and 5- group (C-15) as expected (Fig. 4). The incorporation derived compound 1 in CHCl3 exhibited one signal at ratio was calculated by using FD-MS data by comparing 1.26 ppm, corresponding to deuteriums on the methyl the ½M þ 3þ ion peak (m=z ¼ 295) of the natural and Lasiodiplodin Biosynthesis in Lasiodiplodia theobromae 1121

Fig. 4. 2H-NMR Spectra of Biosynthetically Labeled 1 from 4 (A) and 5 (B), and 1H-NMR Spectrum of Natural 1 (C). A, B) 76.8 MHz, CHCl3. C) 270 MHz, CDCl3.

Fig. 5. Proposed Biosynthetic Pathway to 1. biosynthetically labeled compounds of 1, showing 2H plodin derivative (2) has yet to be revealed; whether enrichment of 0.92% and 0.86% in 1 from 4 and 5, the hydroxylation reaction occurs before the second respectively. NR-PKS reaction or after construction of the lactone This incorporation confirmed the involvement of ring. To elucidate this hydroxylation mechanism, further highly reduced acyl intermediates, namely the 9-hydro- labeling or enzymatic investigation is required. xydecanoyl unit, in the biosynthesis of lasiodiplodin (1). Figure 5 indicates the entire biosynthetic pathway to 1, Materials and Methods showing that five intact acetate units were condensed first by R-PKS to yield a highly reduced pantaketide General. Data were obtained with the following instruments: NMR, acyl intermediate. The acyl chain was next transferred Bruker AMX-500 FT-NMR and Jeol JNM-EX 270 FT-NMR spec- to NR-PKS, which underwent malonyl-CoA condensa- trometers; EI-, FD-, HREI-, and HRFD-MS, Jeol JMS SX-102A mass tion three times, and an aldol condensation to give a spectrometer. The NMR chemical shift values were referenced to 1 resorcylyl intermediate. NR-PKS then catalyzed macro- residual solvent signals as follows: H-NMR, CDCl3 (1H ¼ 7:24); 2 13 lactonization to construct a resorcylic acid lactone H-NMR, CHCl3 (2H ¼ 7:24); C-NMR, CDCl3 (13C ¼ 77:0). skeleton. Finally, O-methylation of the hydroxyl group 2 on C-14 yielded lasiodiplodin as a post-PKS reaction. Preparation of the H-labeled acyl intermediate of 1. 9-Oxononanoic acid (3). Oleic acid (21.5 g, 76.1 mmol) dissolved in To the best of our knowledge, this is the first report dry CH2Cl2 (200 ml) in a flask open to the air was cooled to 78 C on the biosynthesis of 12-membered resorcylic acid while stirring. Ozone was then bubbled into the solution for 3 h until a lactones via the direct incorporation of free fatty acid. faint blue color was observed. Me2S (15.0 ml, 210 mmol) was added, The hydroxylation mechanism for the hydroxyl lasiodi- before the solution was brought to room temperature and then stirred 1122 T. KASHIMA et al.

13 overnight. The solvent containing excess Me2S was then evaporated, Acetylation of 1. To biosynthetically C-acetate-incorporated 1 and the residue was dissolved in EtOAc (100 ml). The solution was (6.6–14.4 mg), pyridine (0.9 ml) and Ac2O (0.3 ml) were added, and washed with brine, dried over Na2SO4, and evaporated to give a the mixed solution was stirred overnight at room temperature. The colorless oil which was purified by silica gel column chromatography reaction mixture was combined with 1 M HCl (15 ml) and extracted (250 g of silica gel, EtOAc:n-hexane = 40:60, v/v) to give 8.83 g of 3 with Et2O(5ml 3). The combined extracts were washed with brine þ as a colorless oil in a 67% yield. HRFD-MS m=z: 173.1169 ½M þ H and dried over Na2SO4. The extract was then concentrated in vacuo to 1 þ (calcd. for C9H17O3, 173.1178); H-NMR (270 MHz, CDCl3) (ppm): give acetylated 1 in a yield of 97–99%. HREI-MS m=z: 334.1771 [M] 1 11.0–9.5 (1H, br s), 9.71 (1H, t, J ¼ 1:8 Hz), 2.38 (2H, td, J ¼ 7:3, (calcd. for C19H26O5, 334.1780); H-NMR (270 MHz, CDCl3) 1.8 Hz), 2.30 (2H, t, J ¼ 7:4 Hz), 1.58 (4H, m), 1.29 (6H, m); (ppm): 6.53 (1H, d, J ¼ 2:0 Hz), 6.49 (1H, d, J ¼ 2:0 Hz), 5.27 13 C-NMR (67.5 MHz, CDCl3) (ppm): 202.9, 180.0, 43.7, 33.9, 28.9, (1H, m), 3.76 (3H, s), 2.70 (1H, dt, J ¼ 13:5, 7.9 Hz), 2.51 (1H, dt, 28.8, 28.7, 24.5, 21.9. J ¼ 13:5, 6.9 Hz), 2.26 (3H, s), 1.91 (1H, m), 1.63 (4H, m), 1.40 (4H, 2 13 [10,10,10- H3] 9-Hydroxydecanoic acid (4). A solution of aldehyde m), 1.34–1.18 (3H, m), 1.30 (3H, d, J ¼ 6:3 Hz); C-NMR (67.5 MHz, 3 (1.50 g, 8.71 mmol) in dry Et2O (30 ml) was cooled to 0 C while CDCl3) (ppm): 169.0, 167.8, 157.1, 151.9, 142.5, 122.6, 114.5, 102.7, stirring, and then treated with a Grignard reagent prepared from Mg 72.3, 56.0, 32.3, 30.4, 29.9, 26.4, 25.4, 24.2, 21.2, 21.1, 19.4. (1.06 g, 43.6 mmol) and CD3I (1.89 ml, 30.0 mmol) in Et2O (20 ml). The reaction mixture was brought to room temperature and left Diacetylation of 2. Biosynthetically 13C-acetate-incorporated 2 overnight, before being treated with a saturated NH4Cl aqueous (2.2–4.7 mg) was diacetylated by Ac2O/pyridine by the same solution (20 ml) and stirred for 30 min. The solution was acidified with procedure as that used to prepare acetylated 1 as just described in a þ 1 M HCl (50 ml) and extracted with Et2O(50 ml 3). The combined yield of 95–99%. HREI-MS m=z: 392.1841 [M] (calcd. for C21H28O7, 1 extracts were washed with brine, dried over Na2SO4, and evaporated to 334.1835); H-NMR (270 MHz, CDCl3) (ppm): 6.53 (1H, d, give a colorless oil. This oil was subjected to silica gel column J ¼ 2:0 Hz), 6.49 (1H, d, J ¼ 2:0 Hz), 5.32 (1H, m), 5.14 (1H, m), chromatography (150 g of silica gel, EtOAc:n-hexane:AcOH = 3.76 (3H, s), 2.80–2.49 (2H, m), 2.26 (3H, s), 2.20 (1H, m), 1.99 (3H, 60:40:1, v/v) to give 1.00 g of 4 as a colorless oil in a 60% yield. s), 1.91 (1H, m), 1.76–1.55 (4H, m), 1.54–1.19 (4H, m), 1.33 (3H, d, þ 13 HRFD-MS m=z: 192.1686 ½M þ H (calcd. for C10H18D3O3, J ¼ 6:6 Hz); C-NMR (67.5 MHz, CDCl3) (ppm): 170.2, 169.0, 1 192.1676); H-NMR (270 MHz, CDCl3) (ppm): 7.10 (1H, br s), 167.5, 157.3, 152.1, 142.3, 122.2, 114.6, 102.8, 69.8, 69.4, 56.0, 36.9, 3.75 (1H, m), 2.28 (2H, t, J ¼ 7:6 Hz), 1.58 (2H, m), 1.38 (2H, m), 31.9, 30.0, 29.9, 24.8, 21.5, 21.3, 21.1, 19.4. 2 1.35–1.22 (8H, m); H-NMR (76.8 MHz, CHCl3) (ppm): 1.09; 13 C-NMR (67.5 MHz, CDCl3) (ppm): 179.3, 68.1, 38.9, 34.0, 29.3, Acknowledgments 29.1, 28.9, 25.5, 24.6. 2 [10,10,10- H3] 9-Hydroxydecanoyl SNAC (5). To a solution of We thank Dr. E. Fukushi and Mr. K. Watanabe for carboxylic acid 4 (589 mg, 3.08 mmol) in DMF (40 ml) at 0 C, measuring the mass spectral and INADEQUATE spec- diphenylphosphoryl azide (1.00 ml, 4.62 mmol) and Et3N (856 ml, tral data. 6.16 mmol) were added. After 2 h of stirring, N-acetylcysteamine (400 ml, 3.70 mmol) was added, and the mixture was stirred at room References temperature overnight. The reaction was quenched with the addition of H2O (150 ml), and the solution was extracted with EtOAc (150 ml 3). 1) Soga S, Shiotsu Y, Akinaga S, and Sharma SV, Curr. Cancer The combined extracts were successively washed with 0.1 M HCl Drug Targets, 3, 359–369 (2003). and brine, and dried over Na2SO4. The extract was concentrated 2) Ninomiya-Tsuji J, Kajino T, Ono K, Ohtomo T, Matsumoto M, in vacuo, and purified twice by silica gel column chromatography Shiina M, Mihara M, Tsuchiya M, and Matsumoto K, J. Biol. 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