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Biosyntheses of alterporriol D and E by Alternaria porri
Phytochemistry, Vol. 30, No. 8, pp. 2593-2595,1991 Printedin Great Britain.
BIOSYNTHESES
003 l-9422/91 $3.00 + 0.00
0 1991PergamonPressplc
OF ALTERPORRIOL
D AND E BY ALTERNARIA
KEIICHIRO OHNISHI, RIKISAKU SUEMITSU, MASAKAZU KUBOTA, HITOSHI MATANO
PORRI*
and YASUMASA YAMADA?
Department of Applied Chemistry, Faculty of Engineering, Doshisha University, Kamikyo-ku, Kyoto 602, Japan; tDepartment of Food Science, Doshisha Women’s College of Liberal Arts, Kamikyo-ku, Kyoto 602, Japan (Receiued in reoisedform 29 January 1991) Key Word Index-Aternaria
poti, biosyntheses; alterporriol D and E; bianthraquinones; pigments.
Abstract-The biosyntheses of alterporriol D and E, metabolites of AItermria porri, were elucidated by incorporation experiments of single and double labelled acetates. ‘“CNMR assignments were also established.
INTRODUCTION
Previously, we reported the polyketide biosynthesis of macrosporin (l), a metabolic pigment of Alternaria porri (Ellis) Ciferri, the causal fungus of black spot disease of stone-leek and onion [l]. We now report the biosyntheses of alterporriol D and E (Ap-D, Ap-E 2) [2], two metabolic pigments and atropisomers of each other of Alternaria porri, utilizing single and double labelled acet-
ates, i.e. [l-“Cl,
[2-13C] and [1,2-‘3C,]. RESULTSAND DISCUSSION
When double labelled acetates were administered, sharp satellite peaks were observed and 13C-13C coupling constants of 2 are given in Tables 1 and 2. The chemical shift of 6 104.9 can be attributable to the aromatic C-7 (Table 1). The chemical shifts at 6 22.4 and 57.1 are apparently attributable to Me-3 and OMe-6. The peaks at 6 165.8 and 166.0 are attributable to aromatic carbons bonded to oxygen, i.e. C-6 and C-8. This is supported by the ’ 3C-1 jC couplings observed between C-5 and C-6 (J = 67.2,67.9 Hz) and C-7 and C-8 (J = 68.6, 69.2 Hz). Other peaks remaining at 6 111.1, 131.8, 143.93 and 143.95 can be attributed to aryl carbons, i.e. C-8a, C5a, C-la and C&I. The peak at 6 111.1, which appeared at a higher magnetic field than those of other angular carbons, can be attributable to C-8a, because C-8a is adjacent to the carbon bearing a hydroxyl group [3]. And the peaks at 6 143.93, 143.95 and 131.8 can be attributed to C-la, C-4a and C-5a, because 13C-13C couplings were observed between C-l and C-la (J=44.4,45.0 Hz), C-4a and C-4 (J =47.7, 47.1 Hz) and C-5a and C-10 (J = 55.8, 55.1 Hz). As the 13C-13C coupling was observed between C-9 and C-8a (J = 57.8,57.8 Hz), the peak at 6 190.7 must be due to C-9 and another carbonyl carbon peak at 6 185.7 must be due to C-10. And the peaks at 6 70.2,70.6, 74.7 and 75.3 are due to alicyclic carbons bonded to oxygens, of which the peak at 6 74.7 can be attributable to C-3, because “C-l 3C coupling was observed C-3 and Me
*part 17 of the series: ‘Studies on the Metabolic Products of Alternaria pod.
For part 16 see ref. @I.
(J-41.441.0 Hz). As mentioned above, 13C-13C couplings were observed between C-l and C-la and G4a and C-4, so peaks at 6 70.6 and 70.2 must be C-l and C-4. The remaining peak at 675.3 must be due to C-2.
2593
2594
K. OHNISHIet Table 1. Incorporation
al.
of sodium [1-‘3C], [2-13C] and [1,2-‘3Cl]acetate D (100 MHz, in CD,OD with TMS) Natural
into alterporriol
C
(ppm)
J 0-W
WI
Enrichment [2-‘“C]Ac [LL3C]Ac
1 la 2 3 4 4a 5 5a 6 7 8 8a 9 10 Me-3 OMe-67
70.6 143.93 75.3 74.7 70.2 143.95 123.6 131.8 165.8 104.9 166.0 111.1 190.7 185.7 22.4 57.1
44.4 45.0 41.0 47.1 47.1 67.2 55.8 67.9 68.6 69.2 57.8 57.8 55.1 41.0 -
80.2 71.2 84.5 89.8 75.2 62.7 44.0 48.9 61.7 78.8 62.7 51.7 46.3 51.7 98.0 100.0
67.8 139.21 123.51 83.1 117.03 _* 92.3: 51.8 65.3 121.31 39.9 85.02 58.5 98.3f 131.q 100.0
131.2$ 68.7 67.5 151.N 54.2 136.6 32.3 85.94 109.3$ 57.6 97.s 32.5 73.q 36.6 88.0 100.0
*When THF was used as a solvent, separated two peaks between la and 4a were observed. tlntensity of signal for OMe was defined as 100%. - No satellite peak. fEnriched with [2-13C]acetate. §Enriched with [1-‘3C]acetate.
Table 2. Incorporation
of sodium [1-‘3C], [2-13C] and [1,2-‘“C,]acetate into alterporriol E (100 MHz, in CD,OD with TMS)
C
(ppm)
J (Hz)
r4
Natural
Enrichment [l-“C]Ac [2-‘“C]Ac
1 la 2 3 4 4a 5 5a 6 7 8 8a 9 10 Me-3 OMe-6’
70.7 144.1 75.2 74.7 70.1 143.8 124.4 131.0 166.7 104.6 166.3 111.0 190.5 185.4 22.4 57.1
44.4 44.4 41.0 47.1 47.1 67.9 55.1 67.9 68.6 69.2 57.8 57.8 55.1 41.0 -
76.9 98.2 57.6 109.8 115.8 95.3 68.5 68.0 78.3 62.8 71.6 61.3 78.6 57.6 118.5 100.0
64.2 141.8t 83.7t 71.6 191.5t 70.8 96.6t 47.1 69.3 101.7t 59.0 91.5t 45.3 79.8t 170.7t 100.0
178.5$ 68.7 55.6 270.0$ 96.9 198.03 34.3 94.21 176.6$ 58.6 139.4$ 55.0 134.1s 53.9 117.9 100.0
*Intensity of signal for OMe was defined as 100%. - No satellite peak. tEnriched with [2-13C]acetate. $Enriched with [1-‘3CJacetate.
Furthermore, to establish the condensation pattern of eight acetate units, sodium [2-13C]- and [l-13C]acetates, were used. In each case, the signal for carbons incorporating 13C on 2 were significantly enriched relative to those
for unlabelled naturally occurring 2 (Table 1). Experimental results for Ap-E are given in Table 2. As Ap-D and E are atropisomers of each other, it is reasonable to find that the values of Ap-E are similar to those of Ap-D,
Biosynthesis of alterporriol D and E by Akernaria porri
2595
For dimeric anthraquinones, there is evidence to indicate that pre-anthraquinones serve as precursors to a number of dimers [S, 63. In the biosyntheses of Ap-D and E, pre-anthraquinones have not been detected by HPLC analysis. Therefore, it might be presumed that Ap-D and E are formed by the oxidative coupling of As-A. EXPERIMENTAL Reagents and apparatus. Na[2-‘%I-, [l-‘“Cl- and [1,2W,]acetate (99 atom% 13C) was purchased from Aldrich. 13CNMR spectra were recorded with a 400 MHz spectrometer.
0
OH
Fungus. The strain of Alternaria porri used was purchased from IF0 Institute for Fermentation, (Osaka), strain no. 9762. Incorporation of single and double lab&d acetates into alterporriol D and E. For the biosynthetic studies, onion deco&ion (150 ml) including labelled acetate (22.5 mg) and unlabelled acetate (51.8 mg) was used as a medium. After fermentation for 3 weeks at 25”, 2 was obtained according to the isolation procedure previously reported [7] and subjected to 1‘C NMR analysis.
I Alterporriol D and E Scheme 1.
except for the chemical shift for C-4a (6 143.8) which appears at a higher magnetic field than that of C-la (6 144.1), the reverse of Ap-D values as shown in Table 1. The combined results show that As-A (3), the monomer component of Ap-D and E, was proved to be derived from octaketide, in which eight acetate units are condensed head to tail, followed by the loss of carboxyl carbon from the terminal unit at C-2, the introduction of a methyl group from methionine [4], and dimerization at C-5 (Scheme 1).
REFERENCES
1. Suemitsu, R., Ohnishi, K., Yanagawase, S., Yamamoto, K. and Yamada, Y. (1989) Phytochemistry 28, 1621. 2. Suemitsu, R., Sakurai, Y., Nakachi, K., Miyoshi, I., Kubota, M. and Ohnishi, K. (1989) Agric. Biol. Chem. 53, 1301. Berger, Y. and Castonguay, A. (1978) Org. Magn. Reson 11, 375.
Threlfall, D. R., Whistance, G. R. and Goodwin, T. W. (1968) Biochem. J. lW, 107. Steglich, W. and Oertel, B. (1984) Sydowia 37, 284. Gill, M. and Steglich, W. (1987) Progr. Ckem Org. Nat. Prod. 51, 1. Suemitsu, R., Sakurai, Y., Nakachi, K., Miyoshi, I., Kubota, M. and Ohnish, K. (1989) Agric. Biol. Clam 53, 1301. Suemitsu, R., Ohnishi, K., Nobuhara, T., Horiuchi, M. and Horiuchi, K. (1990) Agric. Biol. Chem. !!4,2449.
BIOSYNTHESES
003 l-9422/91 $3.00 + 0.00
0 1991PergamonPressplc
OF ALTERPORRIOL
D AND E BY ALTERNARIA
KEIICHIRO OHNISHI, RIKISAKU SUEMITSU, MASAKAZU KUBOTA, HITOSHI MATANO
PORRI*
and YASUMASA YAMADA?
Department of Applied Chemistry, Faculty of Engineering, Doshisha University, Kamikyo-ku, Kyoto 602, Japan; tDepartment of Food Science, Doshisha Women’s College of Liberal Arts, Kamikyo-ku, Kyoto 602, Japan (Receiued in reoisedform 29 January 1991) Key Word Index-Aternaria
poti, biosyntheses; alterporriol D and E; bianthraquinones; pigments.
Abstract-The biosyntheses of alterporriol D and E, metabolites of AItermria porri, were elucidated by incorporation experiments of single and double labelled acetates. ‘“CNMR assignments were also established.
INTRODUCTION
Previously, we reported the polyketide biosynthesis of macrosporin (l), a metabolic pigment of Alternaria porri (Ellis) Ciferri, the causal fungus of black spot disease of stone-leek and onion [l]. We now report the biosyntheses of alterporriol D and E (Ap-D, Ap-E 2) [2], two metabolic pigments and atropisomers of each other of Alternaria porri, utilizing single and double labelled acet-
ates, i.e. [l-“Cl,
[2-13C] and [1,2-‘3C,]. RESULTSAND DISCUSSION
When double labelled acetates were administered, sharp satellite peaks were observed and 13C-13C coupling constants of 2 are given in Tables 1 and 2. The chemical shift of 6 104.9 can be attributable to the aromatic C-7 (Table 1). The chemical shifts at 6 22.4 and 57.1 are apparently attributable to Me-3 and OMe-6. The peaks at 6 165.8 and 166.0 are attributable to aromatic carbons bonded to oxygen, i.e. C-6 and C-8. This is supported by the ’ 3C-1 jC couplings observed between C-5 and C-6 (J = 67.2,67.9 Hz) and C-7 and C-8 (J = 68.6, 69.2 Hz). Other peaks remaining at 6 111.1, 131.8, 143.93 and 143.95 can be attributed to aryl carbons, i.e. C-8a, C5a, C-la and C&I. The peak at 6 111.1, which appeared at a higher magnetic field than those of other angular carbons, can be attributable to C-8a, because C-8a is adjacent to the carbon bearing a hydroxyl group [3]. And the peaks at 6 143.93, 143.95 and 131.8 can be attributed to C-la, C-4a and C-5a, because 13C-13C couplings were observed between C-l and C-la (J=44.4,45.0 Hz), C-4a and C-4 (J =47.7, 47.1 Hz) and C-5a and C-10 (J = 55.8, 55.1 Hz). As the 13C-13C coupling was observed between C-9 and C-8a (J = 57.8,57.8 Hz), the peak at 6 190.7 must be due to C-9 and another carbonyl carbon peak at 6 185.7 must be due to C-10. And the peaks at 6 70.2,70.6, 74.7 and 75.3 are due to alicyclic carbons bonded to oxygens, of which the peak at 6 74.7 can be attributable to C-3, because “C-l 3C coupling was observed C-3 and Me
*part 17 of the series: ‘Studies on the Metabolic Products of Alternaria pod.
For part 16 see ref. @I.
(J-41.441.0 Hz). As mentioned above, 13C-13C couplings were observed between C-l and C-la and G4a and C-4, so peaks at 6 70.6 and 70.2 must be C-l and C-4. The remaining peak at 675.3 must be due to C-2.
2593
2594
K. OHNISHIet Table 1. Incorporation
al.
of sodium [1-‘3C], [2-13C] and [1,2-‘3Cl]acetate D (100 MHz, in CD,OD with TMS) Natural
into alterporriol
C
(ppm)
J 0-W
WI
Enrichment [2-‘“C]Ac [LL3C]Ac
1 la 2 3 4 4a 5 5a 6 7 8 8a 9 10 Me-3 OMe-67
70.6 143.93 75.3 74.7 70.2 143.95 123.6 131.8 165.8 104.9 166.0 111.1 190.7 185.7 22.4 57.1
44.4 45.0 41.0 47.1 47.1 67.2 55.8 67.9 68.6 69.2 57.8 57.8 55.1 41.0 -
80.2 71.2 84.5 89.8 75.2 62.7 44.0 48.9 61.7 78.8 62.7 51.7 46.3 51.7 98.0 100.0
67.8 139.21 123.51 83.1 117.03 _* 92.3: 51.8 65.3 121.31 39.9 85.02 58.5 98.3f 131.q 100.0
131.2$ 68.7 67.5 151.N 54.2 136.6 32.3 85.94 109.3$ 57.6 97.s 32.5 73.q 36.6 88.0 100.0
*When THF was used as a solvent, separated two peaks between la and 4a were observed. tlntensity of signal for OMe was defined as 100%. - No satellite peak. fEnriched with [2-13C]acetate. §Enriched with [1-‘3C]acetate.
Table 2. Incorporation
of sodium [1-‘3C], [2-13C] and [1,2-‘“C,]acetate into alterporriol E (100 MHz, in CD,OD with TMS)
C
(ppm)
J (Hz)
r4
Natural
Enrichment [l-“C]Ac [2-‘“C]Ac
1 la 2 3 4 4a 5 5a 6 7 8 8a 9 10 Me-3 OMe-6’
70.7 144.1 75.2 74.7 70.1 143.8 124.4 131.0 166.7 104.6 166.3 111.0 190.5 185.4 22.4 57.1
44.4 44.4 41.0 47.1 47.1 67.9 55.1 67.9 68.6 69.2 57.8 57.8 55.1 41.0 -
76.9 98.2 57.6 109.8 115.8 95.3 68.5 68.0 78.3 62.8 71.6 61.3 78.6 57.6 118.5 100.0
64.2 141.8t 83.7t 71.6 191.5t 70.8 96.6t 47.1 69.3 101.7t 59.0 91.5t 45.3 79.8t 170.7t 100.0
178.5$ 68.7 55.6 270.0$ 96.9 198.03 34.3 94.21 176.6$ 58.6 139.4$ 55.0 134.1s 53.9 117.9 100.0
*Intensity of signal for OMe was defined as 100%. - No satellite peak. tEnriched with [2-13C]acetate. $Enriched with [1-‘3CJacetate.
Furthermore, to establish the condensation pattern of eight acetate units, sodium [2-13C]- and [l-13C]acetates, were used. In each case, the signal for carbons incorporating 13C on 2 were significantly enriched relative to those
for unlabelled naturally occurring 2 (Table 1). Experimental results for Ap-E are given in Table 2. As Ap-D and E are atropisomers of each other, it is reasonable to find that the values of Ap-E are similar to those of Ap-D,
Biosynthesis of alterporriol D and E by Akernaria porri
2595
For dimeric anthraquinones, there is evidence to indicate that pre-anthraquinones serve as precursors to a number of dimers [S, 63. In the biosyntheses of Ap-D and E, pre-anthraquinones have not been detected by HPLC analysis. Therefore, it might be presumed that Ap-D and E are formed by the oxidative coupling of As-A. EXPERIMENTAL Reagents and apparatus. Na[2-‘%I-, [l-‘“Cl- and [1,2W,]acetate (99 atom% 13C) was purchased from Aldrich. 13CNMR spectra were recorded with a 400 MHz spectrometer.
0
OH
Fungus. The strain of Alternaria porri used was purchased from IF0 Institute for Fermentation, (Osaka), strain no. 9762. Incorporation of single and double lab&d acetates into alterporriol D and E. For the biosynthetic studies, onion deco&ion (150 ml) including labelled acetate (22.5 mg) and unlabelled acetate (51.8 mg) was used as a medium. After fermentation for 3 weeks at 25”, 2 was obtained according to the isolation procedure previously reported [7] and subjected to 1‘C NMR analysis.
I Alterporriol D and E Scheme 1.
except for the chemical shift for C-4a (6 143.8) which appears at a higher magnetic field than that of C-la (6 144.1), the reverse of Ap-D values as shown in Table 1. The combined results show that As-A (3), the monomer component of Ap-D and E, was proved to be derived from octaketide, in which eight acetate units are condensed head to tail, followed by the loss of carboxyl carbon from the terminal unit at C-2, the introduction of a methyl group from methionine [4], and dimerization at C-5 (Scheme 1).
REFERENCES
1. Suemitsu, R., Ohnishi, K., Yanagawase, S., Yamamoto, K. and Yamada, Y. (1989) Phytochemistry 28, 1621. 2. Suemitsu, R., Sakurai, Y., Nakachi, K., Miyoshi, I., Kubota, M. and Ohnishi, K. (1989) Agric. Biol. Chem. 53, 1301. Berger, Y. and Castonguay, A. (1978) Org. Magn. Reson 11, 375.
Threlfall, D. R., Whistance, G. R. and Goodwin, T. W. (1968) Biochem. J. lW, 107. Steglich, W. and Oertel, B. (1984) Sydowia 37, 284. Gill, M. and Steglich, W. (1987) Progr. Ckem Org. Nat. Prod. 51, 1. Suemitsu, R., Sakurai, Y., Nakachi, K., Miyoshi, I., Kubota, M. and Ohnish, K. (1989) Agric. Biol. Clam 53, 1301. Suemitsu, R., Ohnishi, K., Nobuhara, T., Horiuchi, M. and Horiuchi, K. (1990) Agric. Biol. Chem. !!4,2449.