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Porritoxin, a phytotoxin of Alternaria porri
Phyrocheminry, VoL 31.No. 7, PP.2325.-2326, 1992 Printedin Great Britain.
PORRITOXIN,
003l-9422/92 ss.00+ 0.00 0 1992Pergamon Press Ltd
A PHYTOTOXIN
OF ALTERNARZA PORRI
RIKISAKU SUEMITSU, KEIICHIRO OHNISHI, MASAYUKIHORIUCHI, AKIRA KITAGUCHI and Kozo ODAMURA Doshisha University, Kamikyo-ku, Kyoto 602, Japan (Received in revised Jot-m 6 January
1992)
Key Word Index--Alternario porri; Hyphomycetes; phytotoxin; porritoxin; benzoxazocine. Abstract-The culture liquid of Alternaria porri afforded a novel benzoxazocine derivative, porritoxin, whose structure was determined by spectroscopic methods. It inhibited seedling growth in lettuce at a concentration of 10 ppm.
INTRODUCTION
Tentoxin, a non-specific toxin, is produced by Alternaria porri (Ellis) Ciferri, the causal fungus of black spot disease in the stone-leek and onion [l]. Zinniol (1) is also produced by Alternaria porri; this toxin is known additionally in other Akernaria species [2]. This paper deals with the isolation and structural elucidation of a third phytotoxin designated porritoxin (2). Compound 2 represents a new type of benzoxazocine derivative and its inhibitory activities of lettuce and stone-leek roots were also investigated. RESULTS AND DISCUSSION
High resolution mass spectral analysis gave C,,H230,N for 2 and IR absorption was indicative of an amide grouping at 3340, 1670, 1645, methyl and ethyl at 2959 and 1450, ether oxygen at 1125 and 1100, together with a benzene ring at 1600,765 and 700 cn-‘. The UV spectral data showed the existence of a benzoyl group, UV A!$“’ nm (log E) at 215 (4.73). 255 (4.07), 292 (3.60). Compound 2 was optically inactive.
The ‘H and 13CNMR data are given in Table 1. In order to compare them with those of zinniol (l), spectral data of 1 are also shown in Table 1. Comparing the spectral data of 2 with those of 1, the former closely resembles the latter as presented by Starratt [3] and Stoessl [4], except for the disappearance of one of two CH,OH groups in 1 (64.61, 4.69), the remarkable lower shift of an aromatic proton (87.07) (66.66 in l), the existence of two more methylene groups at 63.76 and 3.92, and an amide proton at 63.17. This led us to suspect that 2 is related to 1, su’ch that two CH,OH groups are substituted with CONH and CH,OCH,CHI groups, forming the bicyclic structure as shown in 2. Then, the striking lower shift of an aromatic proton in 2, mentioned above, can be accommodated by the anisotropy of the carbonyl group located at a peri position lo the aromatic hydrogen in 2. Consequently, porritoxin (2) must be 8-(3’,3’-dime~hylallyloxy)-1O-methoxy-9-methyl-1H-3,4dihydro-2,5-benzoxazocin-6(5H)-one. Phytotoxic activities of 2 were also investigated. As shown in the Experimental, the inhibition of root elongation in lettuce and stone-leek seedlings was observed. Table 2 shows that both roots and shoots were weakly affected; thus 2 inhibited the root elongation of both lettuce and stone-leek seedlings by 100% at 40ppm. Meanwhile, 2 inhibited the shoot growth of lettuce seedlings by 9 1.9% at 40 ppm and of stone-leek seedlings by 38.0% at 40 ppm, respectively. As shown in the Table, 2 was found lo inhibit seedling growth at concentrations as low as 10 ppm. The toxicity is greater when compared with other phytotoxic metabolites, e.g. zinniol from Alternmia zinniae which was reported to inhibit the germination of lettuce at 500 ppm [S]. EXPERIMENTAL Fungus. The strain of Alternaria porri used in this experiment was purchased from IF0 (Institute For Fermentation, Osaka), strain no. 9762. Extraction and isolation ofporritoxin. The culture conditions using Richards medium were the same as those previously reported [a]. Alter culturing for 35 days, 10 g of amberlite XAD 7 was added lo 11 of culture liquid, which was stirred overnight. The adsorbates were eluted with Me,CO. The Me&O extract was dissolved in C6H6, washed with 0.1 M NaHCO, and the solvent was evapd lo dryness. The extract was subjected to prep.
2325
R. SUEM~TSUet al.
2326 Table
1. ‘H and “C
NMR spectral
data of porritoxin
(2) and zinniol (I)
Porritoxin* H
(ppm)
1 3 4 5 6 7 8 9 10 11 12 I’ 2 3’ 3’-1 3’-2 Me-9 OMe-IO
4.53 3.92 3.16 3.17 -_ 7.07
s r, J = 4.28 Hz t, J = 4.28 Hz br s
C
(ppm)
I
65.8 62.0 46.5
3 4 6 7 8 9 10 II 12 I’ 2’ 3’ 3’-1 3’-2 Me-9 OMe-10
s
-. 4.57 5.50 . 1.80 1.74 2.20 3.86
Zinniolt
d, J=6.10 Hz t, J = 6.10 Hz s s s s
170.0 101.2 153.4 123.3 158.6 123.9 131.6 50. I 119.6 137.8 18.3 25.8 9.6 59.8
H
(ppm)
C
2-C&OH 2-CH,O_H I-C_H,OH I-CH,Olj
4.61 3.22 4.69 3.22
2-CH,OH I-C_H,OH
6 5 4 3
6.66 s
I’ 2’ 3’ 3’-I 3’-2 Me-4 OMe-3
4SOd, J=7 5.46 1,J=7
s s s s
6 5 4 3 2
1
1.73 1.73 2.14 3.72
Hz Hz
s s s s
I’ 2’ 3’ 3’-1 3’-2 Me-4 OMe-3
(ppm) 65.3 64.3
109.0 157.5 119.8 158.0 124.7 139.0 56.6 119.9 137.5 18.2 25.7 9.2 61.8
l4C@ MHz in CDCI, for ‘H and 100 MHz for 13C NMR. t60 MHz in CDCl, for ‘H” and 50 MHz for “C NMR”.
Table
2. Inhibitory
effects of porritoxin
on lettuce and stone-leek
Rate of inhibition concentration Plant
40 ppm
Lettuce shoot Lettuce root Stone-leek shoot Stone-leek root Control
91.9 (s=O.74) lOO(s=2.16) 38.0 (s = 3.84) 100 (s = 0.63)
seedlings
(%) at a of:
20 ppm
10 ppm
48.6 49.9 31.8 63.9
-
(s =0.88) (s = 4.95) (s=3.20) (s = I. 14)
(s= 1.0) 3.2 (s=6.91) 20.1 (s = 5.25) 23.0 (s = 2.04)
(zero concentration)=0
TLC (Merck Gel 60) in C,H,-Me,CO-HOAc (60:40: 1). The fraction at R, 0.59 was further purified by HPLC using YMC S-343 (Yamamura Chemical Labs) with a solvent of 40% acetonitrile and gave needles of mp 115-I 16” in a yield of 4.2 mg from 8.0 I of the culture medium. Porriroxin. Needles, mp 115-l 16‘; La];’ 0” (MeOH; c 0.5). 1R vz;ccm-‘: 3340 (NH), 2950, 1450 (Me, CH,), 1670, 1645 (CONH), 1600, 765, 700 (phenyl); UV ii$‘” nm (log E): 215 (4.73). 255 (4.07). 292 (3.60). MS (El) m/z: 305.1652 [M] + corresponding to C 17H 230 4N (305.1626). 237.1009 [M+ 1 -C,HP]+ corresponding to C,2H,50,N (237.1000), 222 [237 -Me]‘,206[237-OMe]+,163[206-CONH]’,91 [C,H,]’ and 69 [(Me),C=CHCH,]‘. The ‘H (400 MHz, CDCI,) and 13C (100 MHr CDCI,) NMR data are shown in Table I. Bioassay. Pure sample was dissolved in EtOH and added to distilled H,O with shaking to give a solution of 40 ppm in 2.8% EtOH, from which 20, 10 and 5 ppm solutions were obtained. Each solution (3 ml) was poured onto a piece of filter paper (7 cm diameter) in a Petri dish. Fourteen germinated lettuce seeds (Great Lake) or stone-leek (Kiyotaki) were kept under light (approx. 2ooO Lux) at 25” for 3 days. The average growth of 10
germinated seeds (discarding two upper and lower values) was calculated. As a control, 14 germinated se&s were placed on the filter paper to which only 2.8% EtOH solution was added. Acknowledgemenr --The authors thank Mr K. Matsumura laboratory for his help with the bioassay.
in our
REFERENCES I. Suemitsu,
2. 3. 4. 5. 6.
R., Ohnishi, K., Nobuhara, T., Horiuchi, M. and Horiuchi, K. (1990) Agric. Biol. Chem. 54, 2449. Yu, S., Nishimura, S. and Furuichi, N. (1983) Ann. Phylopalh. Sot. Japan 49, 746. Starratt, A. N. (1968) Can. J. Gem. 46, 767. Stoessl. A., Unwin, C. H. and Stothers, J. B. (1983) Can. J. Chem. 61, 372. White, G. A. and Starratt, A. N. (1967) Can. J. Botany 45, 2087. Suemitsu, R., Horiuchi. K., Horiuchi, M. and Hanabata, M. (1992) Eiosci. Biotech. Biochem. 56, 139.
PORRITOXIN,
003l-9422/92 ss.00+ 0.00 0 1992Pergamon Press Ltd
A PHYTOTOXIN
OF ALTERNARZA PORRI
RIKISAKU SUEMITSU, KEIICHIRO OHNISHI, MASAYUKIHORIUCHI, AKIRA KITAGUCHI and Kozo ODAMURA Doshisha University, Kamikyo-ku, Kyoto 602, Japan (Received in revised Jot-m 6 January
1992)
Key Word Index--Alternario porri; Hyphomycetes; phytotoxin; porritoxin; benzoxazocine. Abstract-The culture liquid of Alternaria porri afforded a novel benzoxazocine derivative, porritoxin, whose structure was determined by spectroscopic methods. It inhibited seedling growth in lettuce at a concentration of 10 ppm.
INTRODUCTION
Tentoxin, a non-specific toxin, is produced by Alternaria porri (Ellis) Ciferri, the causal fungus of black spot disease in the stone-leek and onion [l]. Zinniol (1) is also produced by Alternaria porri; this toxin is known additionally in other Akernaria species [2]. This paper deals with the isolation and structural elucidation of a third phytotoxin designated porritoxin (2). Compound 2 represents a new type of benzoxazocine derivative and its inhibitory activities of lettuce and stone-leek roots were also investigated. RESULTS AND DISCUSSION
High resolution mass spectral analysis gave C,,H230,N for 2 and IR absorption was indicative of an amide grouping at 3340, 1670, 1645, methyl and ethyl at 2959 and 1450, ether oxygen at 1125 and 1100, together with a benzene ring at 1600,765 and 700 cn-‘. The UV spectral data showed the existence of a benzoyl group, UV A!$“’ nm (log E) at 215 (4.73). 255 (4.07), 292 (3.60). Compound 2 was optically inactive.
The ‘H and 13CNMR data are given in Table 1. In order to compare them with those of zinniol (l), spectral data of 1 are also shown in Table 1. Comparing the spectral data of 2 with those of 1, the former closely resembles the latter as presented by Starratt [3] and Stoessl [4], except for the disappearance of one of two CH,OH groups in 1 (64.61, 4.69), the remarkable lower shift of an aromatic proton (87.07) (66.66 in l), the existence of two more methylene groups at 63.76 and 3.92, and an amide proton at 63.17. This led us to suspect that 2 is related to 1, su’ch that two CH,OH groups are substituted with CONH and CH,OCH,CHI groups, forming the bicyclic structure as shown in 2. Then, the striking lower shift of an aromatic proton in 2, mentioned above, can be accommodated by the anisotropy of the carbonyl group located at a peri position lo the aromatic hydrogen in 2. Consequently, porritoxin (2) must be 8-(3’,3’-dime~hylallyloxy)-1O-methoxy-9-methyl-1H-3,4dihydro-2,5-benzoxazocin-6(5H)-one. Phytotoxic activities of 2 were also investigated. As shown in the Experimental, the inhibition of root elongation in lettuce and stone-leek seedlings was observed. Table 2 shows that both roots and shoots were weakly affected; thus 2 inhibited the root elongation of both lettuce and stone-leek seedlings by 100% at 40ppm. Meanwhile, 2 inhibited the shoot growth of lettuce seedlings by 9 1.9% at 40 ppm and of stone-leek seedlings by 38.0% at 40 ppm, respectively. As shown in the Table, 2 was found lo inhibit seedling growth at concentrations as low as 10 ppm. The toxicity is greater when compared with other phytotoxic metabolites, e.g. zinniol from Alternmia zinniae which was reported to inhibit the germination of lettuce at 500 ppm [S]. EXPERIMENTAL Fungus. The strain of Alternaria porri used in this experiment was purchased from IF0 (Institute For Fermentation, Osaka), strain no. 9762. Extraction and isolation ofporritoxin. The culture conditions using Richards medium were the same as those previously reported [a]. Alter culturing for 35 days, 10 g of amberlite XAD 7 was added lo 11 of culture liquid, which was stirred overnight. The adsorbates were eluted with Me,CO. The Me&O extract was dissolved in C6H6, washed with 0.1 M NaHCO, and the solvent was evapd lo dryness. The extract was subjected to prep.
2325
R. SUEM~TSUet al.
2326 Table
1. ‘H and “C
NMR spectral
data of porritoxin
(2) and zinniol (I)
Porritoxin* H
(ppm)
1 3 4 5 6 7 8 9 10 11 12 I’ 2 3’ 3’-1 3’-2 Me-9 OMe-IO
4.53 3.92 3.16 3.17 -_ 7.07
s r, J = 4.28 Hz t, J = 4.28 Hz br s
C
(ppm)
I
65.8 62.0 46.5
3 4 6 7 8 9 10 II 12 I’ 2’ 3’ 3’-1 3’-2 Me-9 OMe-10
s
-. 4.57 5.50 . 1.80 1.74 2.20 3.86
Zinniolt
d, J=6.10 Hz t, J = 6.10 Hz s s s s
170.0 101.2 153.4 123.3 158.6 123.9 131.6 50. I 119.6 137.8 18.3 25.8 9.6 59.8
H
(ppm)
C
2-C&OH 2-CH,O_H I-C_H,OH I-CH,Olj
4.61 3.22 4.69 3.22
2-CH,OH I-C_H,OH
6 5 4 3
6.66 s
I’ 2’ 3’ 3’-I 3’-2 Me-4 OMe-3
4SOd, J=7 5.46 1,J=7
s s s s
6 5 4 3 2
1
1.73 1.73 2.14 3.72
Hz Hz
s s s s
I’ 2’ 3’ 3’-1 3’-2 Me-4 OMe-3
(ppm) 65.3 64.3
109.0 157.5 119.8 158.0 124.7 139.0 56.6 119.9 137.5 18.2 25.7 9.2 61.8
l4C@ MHz in CDCI, for ‘H and 100 MHz for 13C NMR. t60 MHz in CDCl, for ‘H” and 50 MHz for “C NMR”.
Table
2. Inhibitory
effects of porritoxin
on lettuce and stone-leek
Rate of inhibition concentration Plant
40 ppm
Lettuce shoot Lettuce root Stone-leek shoot Stone-leek root Control
91.9 (s=O.74) lOO(s=2.16) 38.0 (s = 3.84) 100 (s = 0.63)
seedlings
(%) at a of:
20 ppm
10 ppm
48.6 49.9 31.8 63.9
-
(s =0.88) (s = 4.95) (s=3.20) (s = I. 14)
(s= 1.0) 3.2 (s=6.91) 20.1 (s = 5.25) 23.0 (s = 2.04)
(zero concentration)=0
TLC (Merck Gel 60) in C,H,-Me,CO-HOAc (60:40: 1). The fraction at R, 0.59 was further purified by HPLC using YMC S-343 (Yamamura Chemical Labs) with a solvent of 40% acetonitrile and gave needles of mp 115-I 16” in a yield of 4.2 mg from 8.0 I of the culture medium. Porriroxin. Needles, mp 115-l 16‘; La];’ 0” (MeOH; c 0.5). 1R vz;ccm-‘: 3340 (NH), 2950, 1450 (Me, CH,), 1670, 1645 (CONH), 1600, 765, 700 (phenyl); UV ii$‘” nm (log E): 215 (4.73). 255 (4.07). 292 (3.60). MS (El) m/z: 305.1652 [M] + corresponding to C 17H 230 4N (305.1626). 237.1009 [M+ 1 -C,HP]+ corresponding to C,2H,50,N (237.1000), 222 [237 -Me]‘,206[237-OMe]+,163[206-CONH]’,91 [C,H,]’ and 69 [(Me),C=CHCH,]‘. The ‘H (400 MHz, CDCI,) and 13C (100 MHr CDCI,) NMR data are shown in Table I. Bioassay. Pure sample was dissolved in EtOH and added to distilled H,O with shaking to give a solution of 40 ppm in 2.8% EtOH, from which 20, 10 and 5 ppm solutions were obtained. Each solution (3 ml) was poured onto a piece of filter paper (7 cm diameter) in a Petri dish. Fourteen germinated lettuce seeds (Great Lake) or stone-leek (Kiyotaki) were kept under light (approx. 2ooO Lux) at 25” for 3 days. The average growth of 10
germinated seeds (discarding two upper and lower values) was calculated. As a control, 14 germinated se&s were placed on the filter paper to which only 2.8% EtOH solution was added. Acknowledgemenr --The authors thank Mr K. Matsumura laboratory for his help with the bioassay.
in our
REFERENCES I. Suemitsu,
2. 3. 4. 5. 6.
R., Ohnishi, K., Nobuhara, T., Horiuchi, M. and Horiuchi, K. (1990) Agric. Biol. Chem. 54, 2449. Yu, S., Nishimura, S. and Furuichi, N. (1983) Ann. Phylopalh. Sot. Japan 49, 746. Starratt, A. N. (1968) Can. J. Gem. 46, 767. Stoessl. A., Unwin, C. H. and Stothers, J. B. (1983) Can. J. Chem. 61, 372. White, G. A. and Starratt, A. N. (1967) Can. J. Botany 45, 2087. Suemitsu, R., Horiuchi. K., Horiuchi, M. and Hanabata, M. (1992) Eiosci. Biotech. Biochem. 56, 139.