Acetylated mycotoxins from Fusarium graminearum

Acetylated mycotoxins from Fusarium graminearum

Phytochemistry, Vol. 28, No. 1, pp. 83-85, 1989. Printedin Great Britain. ACETYLATED 0 MYCOTOXINS L. Mul;roz, J. L. FROM FUSARIUM CASTRO, M. CA...

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Phytochemistry, Vol. 28, No. 1, pp. 83-85, 1989. Printedin Great Britain.




L. Mul;roz,

J. L.




and R.

(x)31-9422/89 $3.00+0.00 1988 PergamonPresspk.


Departamento de Quimica OrgLnica, Universidad de Santiago, Santiago de Compostela, Spain (Receioed in revisedform 26 June 1988) Key Word Index-Fusarium deoxynivalenol,

graminearum; Hyphomycetes; mycotoxins; acetates of zearalenone; cis-zearalenone;

Abstract-In addition to zearalenone, deoxynivalenol and 15-acetyl deoxynivalenol, three new acetylated mycotoxins: 4-acetyl zearalenone, 4-acetyl cis-zearalenone and 3,15-diacetyl deoxynivalenol, were isolated from a Fusarium graminearum cultured on corn. Chemical correlations and relevant spectroscopic data on zearalenone derivatives are presented.


and lb which were further separated and purified by the same technique. Both la and lb presented identical mass spectra and quite similar UV and ‘HNMR spectra indicating that both compounds were monoacetates possessing either the same zearalenone skeleton with a different acetylation pattern at the phenolic groups or a different geometry at the double bond. As no reliable correlation could be established on the basis of existing spectroscopic data [Z], we decided to carry out chemical correlations between 1, and the methylated derivatives of la and lb, i.e. lc and lh, respectively. Selective methylation of 1 produced Id which was acetylated to lf. The other isomer, le, could not be prepared by direct methylation, which produced in all attempts the dimethylated derivative thus contradicting a previous report [3]. Nevertheless, le was prepared by treatment of compound 1 with chloromethyl methyl ether (ClMOM) followed by methylation and deprotection. Acetylation of le gave a compound identical to lc. In order to deduce the substitution pattern of la and lb a series of NOE experiments were performed on lc, If and lh. Thus irradiation of the methoxy group in these three compounds gave NOE with H-3 in lc, with H-3 and H-5 in If and with H-3 in lh. For its part, irradiation of H-2’ produced NOE with H-5, H-5, and H-l’, respectively. These data proved that lc was 4-acetyl-2-methyl zearalenone and so la was 4-acetyl zearalenone. They also suggested a c&skeleton for lh, further supported by the lack of NOE between H-2’ and H-5. In fact, photochemical isomerization [4] of 1, lc, and lf, yielded the three cis-isomers lg, lh and li, respectively, proving that lb was 4-acetyl-cis-zearalenone. Furthermore, submitting lg to the sequence of reactions described for the preparation of lc from 1 (protection, methylation, deprotection and acetylation) the cis-zearalenone derivative lh was obtained. A comparison between the ‘HNMR spectral data obtained for the cis- and trans-zearalenone derivatives showed that the chemical shift for the olefinic proton H-2 was consistently upfield (ca 0.4 ppm) on the cis-compounds and this can be used as a diagnostic signal to distinguish both types of skeletons. The results we present here constitute the first report on the isolation of those acetylated mycotoxins from a

In connection with a survey we are conducting to determine the origin of food rejection by farm animals in Galicia (N.W. Spain), we observed that Fusarium graminearum infected corn was frequently found in fields as well as in stored grain. TLC and HPLC analysis of the toxic corn (hernia salina and rabbit skin test) proved the presence of zearalenone (1) and of deoxynivalenol (2) as the major products, but the recent isolation from the same fungus of 15-acetyl deoxynivalenol, associated with feed refusal by swine [l], prompted us to investigate the production of toxins in a F. graminearum laboratory culture. In this paper we describe the isolation from F. graminearum grown on corn of 4-acetyl zearalenone (la), 4acetyl cis-zearalenone (1 b), 3,15-diacetyl deoxynivalenol (2a) and 15-acetyl deoxynivalenol (2b) [l] in addition to the aforementioned zearalenone (1) and deoxynivalenol (2). Compounds la, lb and 2a have not been isolated previously as natural products. The structures of la and lb have been deduced on the basis of spectroscopic data and chemical correlations. RESULTS AND DISCUSSION

Column chromatography on silica gel of extracts from the mycelium of F. graminearum grown on cracked corn gave three fractions. Fraction C, the most polar one (eluted with hexane-thy1 acetate; 1: 3) produced two compounds further separated by HPLC and identified by comparison [l] of their spectroscopic properties as deoxynivalenol(2) and its 15-acetyl derivative (2b). Fraction B (eluted with hexane-ethyl acetate; 1: 1) produced a compound whose spectroscopic data correspond to 3,15-diacetyl deoxynivalenol (2a). Finally, fraction A, the less polar one (eluted with hexane-ethyl acetate; 2 : 1) showed the NMR spectral characteristics of a zearalenone skeleton. Semifireparative HPLC gave zearalenone (1) as the major product and a mixture of two other faster eluting compounds la

*Author to whom correspondence should be addressed. 83

L. Mul;loz



IfI 1C

Id le If

2 2a 2b


R’ H H Me N Me



Me H



IR lb










natural source. It is also worth noting that lb is the first cis-zearalenone isolated from a fungus. A survey to determine the presence of these compounds on corn samptes is under way. EXPERIMENTAL General. UV were recorded in MeOH soln. NMR spectra were determined on a Bruker WM-250 apparatus in CDCl, soln containing TMS as the int. standard. Mass spectra were obtained on a Kratos MS-25 mass spectrometer by E.I. at 70 eV. HPLC was performed on a Perkin Elmer series 10 chromatograph with a normal phase microporasil column (25 cm x 4.6 mm) eluting with hexane-EtOAc (4: 1). For the toxicity analysis the Artemia sulina [S] and the rabbit skin test [6] were used. Extraction and isolation. To six 1 I erlenmeyer flasks containing 200 g each of sterilized cracked corn (40% humidity) were transferred Fusarium yraminearum inoculums isolated from toxic corn. The flasks were maintained at 27’ in the dark for 15 days and then another 2 weeks at 6’ in the daylight, After that period the mycelium was extracted with Me,CO and the solid residue dried and extracted once with MeCN4% KC1 (9 : 1) and twice with CHCI,. The combined extracts were dried. coned and defatted with hexane-EtOAc (4: 1) finally giving 27 g of an oil which was adsorbed in 15 g of silica gel and submitted to CC on 110 g of the same adsorbent. Elution with hexaneeEtOAc (2 : 1) produced 2.6 g of fraction A which was separated by prep. TLC on silica gel (hexatteeEt,O-HOAc; 35 : I5 : 1). The band coincident with a

et al. sample of zearalenone (1) was isolaied and further submitted to semi-prep. HPLC (hexane-EtOAc; 8 : 2) giving 30 mg of 4-acetyl zearalenone (la) (RR, = 0.671; 15 mg of 4-acetyl-cis-zearalenone lb (RR, =0.86) and 35 mg of zearalenone 1 (RR, =: I ). Elution of the column with hexane~EtOAc (1: 1)gave 1.2 g of a mixture (fraction B) from which was isolated by prep. TLC on silica gel (hexane-EtOAc; 1 : 1) a band (blue fluorescence after spraymg with AICI,) positive in the rabbit skin test which gave 70 mg of 3.15-diacetyl-deoxynivalenol (2a). Finally, elution with hexane-EtOAc (1 : 3) produced Fraction C which after purification by prep. TLC on silica gel (hexane-EtOAc; 1 : 3) gave 40 mg of deoxynivalenol (2) and 20 mg of 15-acctyl deoxynivalenol (2b). Preparution of zeuralenonrdcritatiws lc--1h. Compounds lc and lh were obtained by direct methylation of la and lb with Me,SO, and K,CO, at reflux in dry Me&O. Compound Id was prepared by selective methylation of 1 either by overnight treatment with C’H,N, in an ethereal soln or by reaction with Me,SO, and K,<‘O, in dry Me,CO (20 min at room temp). Compound le was prepared by protection of 1 with chloromethyl methyl ether (1.1 equivalents of NaH and I equivalent of CIMOM in dry THF at room temp), followed by methylation with Me,SO, and deprotectton (stirring with 1 M HCI). The same procedure was used in the preparation of lh from lg. Acetylation of Id and le (Ac,O-pyridine at room temp overnight) gave If and Ic, respectively. Compounds lg-Ii were obtamed by isomerization of the double bond of I. lc and If by a photochemical procedure. In a typical experiment 0.1 mmol of product in IO ml of MeOH were degassed and irradiated with a 400 W mercury lamp for 70 hr. The reaction mixture was concentrated and the cis-isomers separated by prep. TLC. 4-Acetylzeuralenone(la). ‘H NMR (250 MHz. CDCI,): d 1.40 (d, Jz6.2 Hz, 3H, H-l 1’). 1.42-1.85 and 2042.42 (m, 10H). 2.30 (s, 3H, OAc), 2.6O(m, LH, H-7’), 2.82 (at, lH, H-5’), 5.03 (m, lH, Hlo’), 5.72(m, lH, H-2’),6.66(1, J-2.7 Hz,ZH, H-3 and H-5), 7.02 (dd, J=1.6Hz and J=15.3Hz, IH, H-l’), 11.91 (s, 1H. OH). 13CNMR (62.X3 MHz., CDCI,): 6210.93 (C-6’). 171.12 (C-12’). 168.70 (OAc), 164.61 (C-2) 155.01 (C-4), 143.36 (C-6). 133.48 (Cl’), 132.64 (C-2’), 113.65 (C-3), 109.57 (C-5). 108.30 (C-l), 74.03 (ClO’), 42.87 (C-7’). 36.64 (C-5’). 34.64 (C-9’), 30.99 (C-3’). 22.04 (C8’) 21.04 (OACJ. 20.95 (C-4) 20.63 (C-l 1’). UV j.“,::” nm: 230, 264, 322. MS m;z (rel. in(.): 360 [M] + (3X), 300 (14). 249 (13) 246 (8) 231 (14). 206 (33). 1X8 (loo), 151 (24). 125 (24) 112 (35). 4-Acetyl-2-mrthyl :earalenone (1~). ‘H NMR (250 MHz, CDCl,):61.33(d. J-6.3 Hz.3H, H-II’). [email protected], 12H),2.31 (s, 3H, OAc), 3.81 (s. 3H, OMe). 5.33 (m, 1H. H-IO’). 6.02(m. 1H. H-2),6.33 (d,J ==15.6 Hz. lH,H-l’), 656(d,J= 1.8 Hz, lH,H-3), 6.85 (d, J= 1.X Hz, lH, H-5). ‘.‘CNMR (62.83 MHz, CDCI,): a211.40(C-6’). 169.18 (C-12’), 167.101OAc). 157.31 (C-2). 152.23 (C-4), 136.77 (C-6) 134.24 (C-l’). 128.16 (C-2’). 110.49 (C-3). 105.43 (C-l), 103.88 (C-5). 71.49(C-IO’). 56.14(OMe)44.03(C-7’) 37.55 (C-5’), 35.07 (C-9’) 31.16 (C-3’). 21.66 (C-8’) 21.20 (C-4’), 21.04 (OAc), 19.93 (C-l 1’). 4-Methyl zcaralerione (Id). ‘H NMR (250 MHz. CDCI,): 61.39 (d, .J=6.2 Hz. 3H. H-11’), 1.51 (m. lH, H-4’), 1.61-1.83 (m, 4H, H-X’ and H-9’) 2.06-2.24 (m, 4H). 2.39 (m. lH, H-3’), 2.62 (m, lH, H-7’), 2.85 (m. IH, H-5’) 3.82 (s. 3H, OMe), 5.02 (m, lH, H10’) 5.6X (m, lH, H-2’), 6.39 (d, J=2.7 Hz, IH. ArH), 6.45 (d. J =2.7 Hz, lH, ArH). 7.02 (dd, J== 15.3 Hz and J=2.0 Hz, 1H. H1’). 2-M&y/ zeuralenonr (le). ‘H NMR (250 MHz, CDCI,): S1.34(d, J=6,3Hz,3H,H-11’): 1.45.-?.50(m, llH),273(m, IH, H-5’), 3.77 (s, 3H, OMe), 5.31 (m, lH, H-10’). 5.95 (m, lH, H-2’), 6.33(d.J= 152Hz, IH.H-1’).6.33(s. lH.ArH),6.53(~, lH,ArH).


Mycotoxins from Fusarium graminearum 2-Acetyl-4-methyf zearalenone (If). ‘H NMR (250 MHz, CDCI,):61.31 (d,J=6.3 Hz, 3H,H-11’), 1.55-2.42(m, llH),2.27 (s, 3H, OAc), 2.64 (m, lH, H-S), 3.83 (s, 3H, OMe), 5.26(m, lH, HlO’), 5.95 (m, lH, H-2’), 6.55 (d, J= 15.6 Hz, lH, H-l’), 6.56 (d, J =2.4 Hz, lH, ArH), 6.89 (d, J=2.4 Hz, lH, ArH). ‘%NMR (62.83 MHz, CD&): 6211.28 (C-6’), 168.99 (C-12’), 165.99 (OAc), 161.14 (C-4), 157.60 (C-2), 138.63 (C-6), 133.98 (C-l’), 129.33 (C-2’), 118.45 (C-l), 109.32 (C-3), 107.48 (C-5), 71.36 (ClO’), 55.55 (OMe) 43.65 (C-7’), 37.42 (C-5’), 35.06 (C-9’), 31.29 (C3’), 21.48 (C-8’). 21.15 (C-4’), 20.76 (OAc), 19.75 (C-11’). cis-Zearalenone (lg). ‘H NMR (250 MHz., CDCI,): 6 1.34 (d, J =6.3 Hz, 3H, H-11’), 1.44-2.31 (m, llH), 2.53 (m, lH, H-5’), 5.14 (m, lH, H-10’), 5.41 (m, lH, H-2’), 6.20 (d, J=2.2 Hz, lH, ArH), 6.39 (d, J=2.2Hz, lH, ArH), 6.63 (d, J=l2.0 Hz, lH, H-l’). ‘%NMR (62.83 MHz, CDCI,): 6212.16 (C-6’), 171.04 (C-12’), 165.48 (C-2), 160.84 (C-4), 142.14 (C-6), 131.96 (C-l’), 129.98 (C2’), 111.54 (C-3), 104.33 (C-l), 102.28 (C-5), 73.71 (C-10’), 41.63 (C7’), 40.71 (C-5’), 33.01 (C-9’), 28.83 (C-3’), 21.25 (C-8’, C-4’, C-l 1’). 4-Acetykis-zearalenone (lb). ‘H NMR (2.50 MHz, CDCl,): 61.36 (d, J=6.3 Hz, 3H, H-11’), 1.45-2.62 (m, 12H), 2.30 (s, 3H, OAc), 5.15 (m, lH, H-10’), 5.46 (m, lH, H-2’), 6.41 (d, J=2.5 Hz, lH, ArH), 6.63 (dd, J=2.1 Hz, J= 12.1 Hz, IH, H-l’), 6.68 (d, J =2.5 Hz, lH, ArH). 13C NMR (62.83 MHz, CDCI,): b210.26 (C6’), 170.74(C-12’), 168.56 (OAc), 164.7O(C-2), 154.55 (C-4), 141.61 (C-6), 131.12 (C-l’), 131.00 (C-2’), 120.83 (C-3), 109.54 (C-l), 109.43 (C-5), 74.47 (C-lo’), 41.52 (C-7’), 40.68 (C-5’), 30.02 (C-9’), 28.87 (C-3’), 21.29 (C-S’), 21.20 (OAc), 21.15 (C-4’), 21.04 (C-l 1’). UVI.f:p nm: 219, 253, 315. MS m/z (rel. int.): 360 [M]’ (44), 300 (13), 249 (12), 246 (15), 231 (16), 206 (27), 188 (lOO), 151 (25), 125 (24), 112 (38). 4-Acetyl-2-methyl cis-zeara[enone (lb). ‘H NMR (250 MHz, CDCI,): 61.37(d, J=6.3 Hz, 3H, H-11’), 1.42-2.75 (m, 12H), 2.36 (s, 3H, OAc), 3.82 (s, 3H, OMe), 5.21 (m, lH, H-10’), 5.55 (m, lH, H-2’), 6.36 (dd, J=2.4 Hz, J=lO.2 Hz, lH, H-l’), 6.57 (d, J =2.0 Hz, lH, ArH), 6.59 (d, J=2.0 Hz, lH, ArH). 2-Acetyl-Cmethyl cis-zearalenone (li). ‘H NMR (250 MHz, CDCI,): 61.33 (d, J = 6.3 Hz, 3H, H-11’), 1.562.42 (m, 12H),

2.30 (s, 3H, OAc), 3.82 (s, 3H, OMe), 5.17 (m, lH, H-10’), 5.55 (m, lH, H-2’), 6.56(d, J=ll.6Hz, lH,H-1’),6.57(d, J=2.1 Hz, lH, ArH), 6.63 (d, J=2.1 Hz, lH, ArH). 15-dcetyl deoxyniualenol (Zb). ‘HNMR (250 MHz, CDCI,): 61.10 (s, 3H, 14-Me), 1.90 (s, 3H, OAc), 1.91 (dd, J=O.8 Hz, J =1.5Hz,3H, 16-Me),2.13(dd, J=l0.6Hz, J=l4.8Hz, lH, H4),2.25 (dd, J=4.5 Hz, J=l4.8 Hz, lH, H-4), 3.11 (d, J=4.2Hz, lH, H-13), 3.16(d, J=4.2 Hz, lH, H-13), 3.67 (d, J=4.5 Hz, lH, H-2), 3.77(d, J=2.0 Hz, 7-OH),4.26(s,2H, H-15),4.57(m, lH, H3), 4.86 (d, J=2.0Hz, lH, H-7), 4.91 (d, J=5.9Hz, lH, H-11), 6.63 (m, J = 1.5 Hz, J = 5.9 Hz, lH, H-10). 3,15-Diacetyl-deoxyniualenol @I). ‘H NMR (250 MHz, CDCI,): 61.12 (s, 3H, 14-Me), 1.90 (s, 6H, 16-Me, 15-OAc), 2.15 (s, 3H, 3-OAc), 2.19 (dd, J=ll.OHz, J=l5.2Hz, lH, H-4), 2.33 (dd, J=4.6 Hz, J= 15.2 Hz, lH, H-4), 3.13 (d, J=4.2 Hz, lH, H-13), 3.17(d,J=4.2Hz,lH,H-l3),3.79(d,J=l.8H~lH,7-OH),3.93 (d,J=4.4Hz,1H,H-2),4.25(d,J=l2.0Hz,lH,H-l5),4.29(d,J = 12.0 Hz, lH, H-15), 4.712 (d, J=5.8 Hz, lH, H-11), 4.83 (d, J =1.8 Hz, lH, H-7), 5.24 (m, J=4.6Hz, J=lO.9 Hz, lH, H-3), 6.59 (m, J=l.5 HZ, J=5.8 Hz, lH, H-10). Acknowledgements-We thank the Comisibn Cientifica Asesora de la Xunta de Galicia for financial support. L. M. is grateful to the Diputaci6n Provincial de la Coruiia for a grant. REFERENCES 1. Abbas, H. K., Mirocha, C. J. and Tuite, J. (1986) Appl. Environ. Microbial. 51, 841. 2. El-Sharkawy, S. H. and Abul-Hajj, Y. J. (1988), J. Org. Chem. 53, 515. 3. Urry, W. H., Wehrmeister, H. L., Hodge, E. B. and Hidy, P. H. (1966) Tetrahedron Letters 21, 3109. 4. Peters, C. A. (1972) J. Med. Chem. 15, 867. 5. Eppley. R. M. (1974) J. A.O.A.C. 57, 618. 6. Chung, H. W., Trucksess, M. W., Giles, A. L. and Frieman, D. (1974) J. A.O.A.C. 57, 1120.