- Email: [email protected]
Botryslactone, a new plant growth regulator produced by Botrytis squamosa
Terrahedron
L&m,
Pergamon
Vol. 36. No. 42, pp. 1613-7616, 1995 Elsevier Science Ltd Printed in Great Britain oD404039/95
$9.50+0.00
0040.4039(95)01609-O
Botryslactone, A New Plant Growth Regulator Produced by Botrytis squamosa Yasuo Kimura* + , Hiroaki Fujioka*,
Takashi Hamasaki*, Kazuo Furihata**,
and Shozo Fujioka***
* Department of Bioresource Science, Tottori University, Koyama, Tottori 680, Japan ** Laboratory of NMR, The University of Tokyo, Bunkyou-ku, Tokyo 113, Japan *** The Institute of Physical and Chemical Research, Wako-shi, Saitama 351-01, Japan
Abstract: repulntcx
Botryslactone (1) was ~wlatel from the mtabolite and tts structure was estabbshtxl by NMR studm.
of Borryk
~quamosaa
as a new plant
growth
Borrytis squumosa is a well-known pathogen which causes gray-molt neck rot in Allium cepa L.(onion), small sclerotialneck rot in AlfiumchitwnseG. Don (scallion),and gray-mold leaf blight in Album fubersosum Rotter (Chinese chive). In the course of our search for plant growth regulators in metabolites of this fungus, we have isolated deacetyldihydrobotrydial,l) 0-methyldihydrobotrydiah2) deac+Ometbyldihydrobotrydialone,2) BSF-A3) together with dehydrobotrydienaJ4) as a known compound. In our continuing research for new biologically active substances. we found a new hypocotyl growth-inhibiting and rootpromoting substance, which was named botryslactone (1). Here we describe the structural elucidation and brief biological activities of 1. The fungus, Botryis squumosu , was cultured stationarily in a malt extract medium at 24 “C for 21 days. The culture filtrate was treated with active charcoal at pH 2.0, and then successively extracted twice with acetone. The combined solvents were concentrated in vucuo, and the residue was chromatographed on a silica gel and Sephadex LH-20 column. A multistep fractionation by chromatography afforded botryslactone (1) as colorless plates (mp 77. 0 “C ) in a yield of 0.9 mgn The molecular formula of L was established as ClgH2404
CH3 CH3 12
1
13
by HREJ-MSI(M +) , m/z 304.1615 (-0.7 mmu error)]. tH and ‘k data of 1 are shown in the Table. Since the UV spectrum of 1 0. max EtOH (E); 220 (sh. SSSO), 271 (8470), 278 (8280) was similar to that of a reduced dehydrobotrydiena14) by NaBH45) and four methyl signals at 8H 2.27 (C-l I), 1.28 (C-
12, C-13) and 1.46 (C-14) were observed, the presence of a 2.6,6,8- tetramethyl 1,8-disubstituted indan moiety (A) in 1 was suggested. Furthermore, the aromatic signals at ?)H 7.06 (C-3) and 7.08 (C-4) were
7674
assigned to be orrho protons by coupling constant (J=7.0). The signals at 6H 1.75 (1H) and 2.38 (1H) showinggemid coupling of J= I 1.5 Hz indicated the methylene protons at C-7. The IR absorption band at 3370 cm-l and the signals at 6H 3.68 (lH, dd, J=4.6, 9.0 Hz), 3.72( IH, dd, 5=4.6, 9.0 Hz), and 3.98 (lH, dd, k4.0, 4.6 Hz) indicated the presence of a hydroxylmethyl group (B) in 1. This observation was further supported by the fact that the signals at 8H 3.98 as well as 5.30 disappeared on addition of D20, and the signals at ?JH 3.68 and 3.72 were shifted at bH 4.16 (2H, br s) in the IH-NMR spectrum of a diacetylated compound6) of 1 by Ac2O/pyridine, and the peak at m/z 274 (M+-30) appeared as a prominent one in MS of 1. This hydroxymethyl group should be connected to C-8 in (A) because of the chemical shift of metbylene protones (Cl5H) at 6H 3.68 and 3.72. HMBC correlations between 15-H and C8, C-7, and C-9 further indicated this bonding. The signal at 6H 4.52 (C-1’7H) was coupled to the methylene protones at ?jH 2.55 and 2.65,which were subsequently coupled to the methine proton at BH 6.30. In the IH-NMR spectrum of a diacetylated compound,6) the signal at 6H 4.52 was shifted lower field at 6H 5.30. This datum showed that a hydroxy group was attached to C-17. On the basis of these results and IR absorption band at 1765 cm-l as well as the remaining G$H403 obtained by deducting the partial structures (A and B) from the molecular formula, a partial structure(C) having a y-lactone moiety was derived.
I--H~OH H
H
m Thus. C-10 was connected to C-l of (.4). This result was also supported by HMBC correlations between 10-H and C- I, C 2. and C-9. From these results, the plane structure of 1 was established, and confirmed by HMBC spectrum. The results of HMBC and NOESY experiments are shown in Figs. The stereochemistry at C-17 in 1 was determined by the HOICW method,1 ) in which the y-lactone has a secondary alcohol. Since treatment of I with (-t )-a- phenylbutyric anhydride liberated (+)-a-phenylbutyric acid, the absolute stereochemistry at C-17 is R. Also, negative Cotton effect 8)at 230 nm (A E=-2.86. c 0.007, EtOH) supported the absolute stereochemistry at C-17 to be R. In the NOESY spectrum of 1, C-17H and C-1OH is ~?WZS.Thus, the absolute stereochemistry of C-10 is S. The absolute stereochemistry at C-8 of 1 should possess the same stereochemistry from the biosynthetic considerations of the metabolites such as deacetyldihydrobotrydial, ’ ) 0methyldihydrobotrydia1, 2, deacetayl O-methyldihydrobotrydialone, 2)dehydrobotrydienal,4) botrydienal,4) deac&ylbo@ydial,4) of B. sqrcamosn. Thus, the methyl group (C-14) is aoriented. Botryslactone ( 1 I was examined for the activity toward the growth of letuce seedlings.9) The hypocotyl growth was inhibited in proportion to the concentrations of 1 and IC50 was 3oOmg/l . On the other hand, the root growth was promoted in proportion
to the concentrations between l-300 mg/l of 1 and the
growth was accelerated by 180 % at the concentration of 300 mg/l. However, 1 inhibited the root growth at the concentration of 1000 mgll. Biological activities of the metabolites obtained from B. squumosu , including 1, will be mported in detail m the near future.
7675
HMBC
1H (500 MHz)
Carbon Number
13c
1 2 3 4 5 6
134.2 136.6 135.5 124.7 153.2 41.7
7
55.0 (t)
8 9 10 11
51.6 144.6 78.6 21.2
of 1
experiments
(s) (s) (d) (d) (s) (s)
(s) (s) (d) (q)
and 13C (125 MHz) IH
7.06 (1 H, d, 7.0) 7.08 (lH, d, 7.0)
1.75 (Ha, d, 11.5) 2.38 (Hg), d, 11.5)
NOESY experiments of 1
NMR
Data for
Carbon Number
13C
12 13 14 15
31.9 33.0 26.7 71.9
(q)
16
38.7 (t)
(9)
(q) (t)
17 69.9 td) 18 177.5 (s) 15-OH 17-OH
1 in CD3COCD3
1H
1.28 1.28 1.46 3.68 3.72 2.65 2.55 4.52
(3H, s) (3H, s) (3H, s) (lH, dd, (lH, dd, (Ha,ddd, (HS,ddd, (lH, dd,
4.6, 9.0) 4.0, 9.0) 6.1, 8.5,13.5) 1.4, 6.1,13.5) 1.4, 6.1)
3.98 (IH, dd, 4.0,4.6) 5.30 (lH, br s)
6.30 (lH, dd, 6.1, 8.5) 2.27 (3H, s)
The s, d, t, and q in parenthese of 13C NMR spectrum showed multiplicities determined by INEPT experiments. Also the s and d, and the numbers in parenthes of 1H NMR spectrum showed multiplicities and coupling constants, respectively.
7676
HEFEHENCES
1.
and NOTES
Kimura, Y.; Fujioka, H.; Nakajima, H.; Hamasaki, T.; Irie, M.; Fukuyama, K.; Isogai, A. Agric. Bid. Chem, 1986, SO, 2123-2125.
2.
Kimura, Y.; Fujioka, H.; Nakajima, H.; Hamasaki, T.; Isogai, A. Agric. Biol. Chem,1988,
52.
18451847. 3.
Kimura, Y.; Fujioka. H.; Nakajima, H.; Hamasaki, T.; R. Nakashima, R. Biosci.Biotech.B&hem, 1993, 57, 1584 -1585.
4.
Kimata, T.; Natsume, M.; Marumo, S. Terruhedron
5.
The physicochemical
2940,2900,2500,
1480, 1460, 1380, 1200, 1050, 1015,
UV h max EtoH nm (6): 220 (sh. 1l,lOO), 271 (2020), 279 (1980). EIMS m/z: (rel.
intensity): 234 ]M’]
(1 l), 203(49), 187( 100). 172(68), 157(49). lH-NMR
(6H, s), 1.32 (3H. s) 1.65 (lH, d, 5=14.0 Hz), 2.05 (lH,d, 5=12.0 Hz), 3.85 (lH, d, &12.0 6.
26, 2097-2100.
properties of this compound are as follows. Colorless amorphous.
IR v max Fi1m cm -l: 3350,3250,2980, 950,835.
1985,
L&t.
The physicochemical 2970,2945,2890,
(TMS, CD3COCD3):
1.20
J=14.0 Hz), 2.38 (3H, s), 3.63 (lH, d,
Hz), 4.70 (2H, s), 7.00 (2H, s).
properties of this compound are as follows. Colorless oil. IR v max Film cm -1: 1705, 1750, 1460, 1380, 1260, 1240, 1210, 1060, 1040,910,810.
UV A
max EtCH nm (4: 271 (2230). 279 (2330). EIMS m/z: (rel. intensity): 388]M+J (lo), 329(14), 315(70), 256(54), 228(12), 199(16), 185(20). lH-NMR
(TMS CDC13): 1.29 (6H, s), 1.52 (3H, s)
1.83 (lH, d, 5=14.0 Hz),2.02 (3H. s), 2.18 (3H, s), 2.27 (3H, s), 2.40 (lH, d, J=14.0 Hz), 2.55 (2H, m), 4.16 (2H, br s), 5.35 (lH, br dd, 5=3.5, 8.0 Hz), 6.23 (lH, br t, 3=8.0 Hz), 7.10 (2H, s). 7.
Horeau, A.; Kagan, H. B. Tad&on,
8.
Okuda, T; Harigaya, S; Kiyomoto,
9.
Frankland, B.; Wareing,P. F. NATURE, 1960,
(Received
in Japan
17 July
1995; revised
1964,
20, 243 l-2441.
A. Chem.Pharm Bull. 1964,
21 August
i2,
304-306.
18.5. 255-256.
1995; accepted
25 August
1995)
L&m,
Pergamon
Vol. 36. No. 42, pp. 1613-7616, 1995 Elsevier Science Ltd Printed in Great Britain oD404039/95
$9.50+0.00
0040.4039(95)01609-O
Botryslactone, A New Plant Growth Regulator Produced by Botrytis squamosa Yasuo Kimura* + , Hiroaki Fujioka*,
Takashi Hamasaki*, Kazuo Furihata**,
and Shozo Fujioka***
* Department of Bioresource Science, Tottori University, Koyama, Tottori 680, Japan ** Laboratory of NMR, The University of Tokyo, Bunkyou-ku, Tokyo 113, Japan *** The Institute of Physical and Chemical Research, Wako-shi, Saitama 351-01, Japan
Abstract: repulntcx
Botryslactone (1) was ~wlatel from the mtabolite and tts structure was estabbshtxl by NMR studm.
of Borryk
~quamosaa
as a new plant
growth
Borrytis squumosa is a well-known pathogen which causes gray-molt neck rot in Allium cepa L.(onion), small sclerotialneck rot in AlfiumchitwnseG. Don (scallion),and gray-mold leaf blight in Album fubersosum Rotter (Chinese chive). In the course of our search for plant growth regulators in metabolites of this fungus, we have isolated deacetyldihydrobotrydial,l) 0-methyldihydrobotrydiah2) deac+Ometbyldihydrobotrydialone,2) BSF-A3) together with dehydrobotrydienaJ4) as a known compound. In our continuing research for new biologically active substances. we found a new hypocotyl growth-inhibiting and rootpromoting substance, which was named botryslactone (1). Here we describe the structural elucidation and brief biological activities of 1. The fungus, Botryis squumosu , was cultured stationarily in a malt extract medium at 24 “C for 21 days. The culture filtrate was treated with active charcoal at pH 2.0, and then successively extracted twice with acetone. The combined solvents were concentrated in vucuo, and the residue was chromatographed on a silica gel and Sephadex LH-20 column. A multistep fractionation by chromatography afforded botryslactone (1) as colorless plates (mp 77. 0 “C ) in a yield of 0.9 mgn The molecular formula of L was established as ClgH2404
CH3 CH3 12
1
13
by HREJ-MSI(M +) , m/z 304.1615 (-0.7 mmu error)]. tH and ‘k data of 1 are shown in the Table. Since the UV spectrum of 1 0. max EtOH (E); 220 (sh. SSSO), 271 (8470), 278 (8280) was similar to that of a reduced dehydrobotrydiena14) by NaBH45) and four methyl signals at 8H 2.27 (C-l I), 1.28 (C-
12, C-13) and 1.46 (C-14) were observed, the presence of a 2.6,6,8- tetramethyl 1,8-disubstituted indan moiety (A) in 1 was suggested. Furthermore, the aromatic signals at ?)H 7.06 (C-3) and 7.08 (C-4) were
7674
assigned to be orrho protons by coupling constant (J=7.0). The signals at 6H 1.75 (1H) and 2.38 (1H) showinggemid coupling of J= I 1.5 Hz indicated the methylene protons at C-7. The IR absorption band at 3370 cm-l and the signals at 6H 3.68 (lH, dd, J=4.6, 9.0 Hz), 3.72( IH, dd, 5=4.6, 9.0 Hz), and 3.98 (lH, dd, k4.0, 4.6 Hz) indicated the presence of a hydroxylmethyl group (B) in 1. This observation was further supported by the fact that the signals at 8H 3.98 as well as 5.30 disappeared on addition of D20, and the signals at ?JH 3.68 and 3.72 were shifted at bH 4.16 (2H, br s) in the IH-NMR spectrum of a diacetylated compound6) of 1 by Ac2O/pyridine, and the peak at m/z 274 (M+-30) appeared as a prominent one in MS of 1. This hydroxymethyl group should be connected to C-8 in (A) because of the chemical shift of metbylene protones (Cl5H) at 6H 3.68 and 3.72. HMBC correlations between 15-H and C8, C-7, and C-9 further indicated this bonding. The signal at 6H 4.52 (C-1’7H) was coupled to the methylene protones at ?jH 2.55 and 2.65,which were subsequently coupled to the methine proton at BH 6.30. In the IH-NMR spectrum of a diacetylated compound,6) the signal at 6H 4.52 was shifted lower field at 6H 5.30. This datum showed that a hydroxy group was attached to C-17. On the basis of these results and IR absorption band at 1765 cm-l as well as the remaining G$H403 obtained by deducting the partial structures (A and B) from the molecular formula, a partial structure(C) having a y-lactone moiety was derived.
I--H~OH H
H
m Thus. C-10 was connected to C-l of (.4). This result was also supported by HMBC correlations between 10-H and C- I, C 2. and C-9. From these results, the plane structure of 1 was established, and confirmed by HMBC spectrum. The results of HMBC and NOESY experiments are shown in Figs. The stereochemistry at C-17 in 1 was determined by the HOICW method,1 ) in which the y-lactone has a secondary alcohol. Since treatment of I with (-t )-a- phenylbutyric anhydride liberated (+)-a-phenylbutyric acid, the absolute stereochemistry at C-17 is R. Also, negative Cotton effect 8)at 230 nm (A E=-2.86. c 0.007, EtOH) supported the absolute stereochemistry at C-17 to be R. In the NOESY spectrum of 1, C-17H and C-1OH is ~?WZS.Thus, the absolute stereochemistry of C-10 is S. The absolute stereochemistry at C-8 of 1 should possess the same stereochemistry from the biosynthetic considerations of the metabolites such as deacetyldihydrobotrydial, ’ ) 0methyldihydrobotrydia1, 2, deacetayl O-methyldihydrobotrydialone, 2)dehydrobotrydienal,4) botrydienal,4) deac&ylbo@ydial,4) of B. sqrcamosn. Thus, the methyl group (C-14) is aoriented. Botryslactone ( 1 I was examined for the activity toward the growth of letuce seedlings.9) The hypocotyl growth was inhibited in proportion to the concentrations of 1 and IC50 was 3oOmg/l . On the other hand, the root growth was promoted in proportion
to the concentrations between l-300 mg/l of 1 and the
growth was accelerated by 180 % at the concentration of 300 mg/l. However, 1 inhibited the root growth at the concentration of 1000 mgll. Biological activities of the metabolites obtained from B. squumosu , including 1, will be mported in detail m the near future.
7675
HMBC
1H (500 MHz)
Carbon Number
13c
1 2 3 4 5 6
134.2 136.6 135.5 124.7 153.2 41.7
7
55.0 (t)
8 9 10 11
51.6 144.6 78.6 21.2
of 1
experiments
(s) (s) (d) (d) (s) (s)
(s) (s) (d) (q)
and 13C (125 MHz) IH
7.06 (1 H, d, 7.0) 7.08 (lH, d, 7.0)
1.75 (Ha, d, 11.5) 2.38 (Hg), d, 11.5)
NOESY experiments of 1
NMR
Data for
Carbon Number
13C
12 13 14 15
31.9 33.0 26.7 71.9
(q)
16
38.7 (t)
(9)
(q) (t)
17 69.9 td) 18 177.5 (s) 15-OH 17-OH
1 in CD3COCD3
1H
1.28 1.28 1.46 3.68 3.72 2.65 2.55 4.52
(3H, s) (3H, s) (3H, s) (lH, dd, (lH, dd, (Ha,ddd, (HS,ddd, (lH, dd,
4.6, 9.0) 4.0, 9.0) 6.1, 8.5,13.5) 1.4, 6.1,13.5) 1.4, 6.1)
3.98 (IH, dd, 4.0,4.6) 5.30 (lH, br s)
6.30 (lH, dd, 6.1, 8.5) 2.27 (3H, s)
The s, d, t, and q in parenthese of 13C NMR spectrum showed multiplicities determined by INEPT experiments. Also the s and d, and the numbers in parenthes of 1H NMR spectrum showed multiplicities and coupling constants, respectively.
7676
HEFEHENCES
1.
and NOTES
Kimura, Y.; Fujioka, H.; Nakajima, H.; Hamasaki, T.; Irie, M.; Fukuyama, K.; Isogai, A. Agric. Bid. Chem, 1986, SO, 2123-2125.
2.
Kimura, Y.; Fujioka, H.; Nakajima, H.; Hamasaki, T.; Isogai, A. Agric. Biol. Chem,1988,
52.
18451847. 3.
Kimura, Y.; Fujioka. H.; Nakajima, H.; Hamasaki, T.; R. Nakashima, R. Biosci.Biotech.B&hem, 1993, 57, 1584 -1585.
4.
Kimata, T.; Natsume, M.; Marumo, S. Terruhedron
5.
The physicochemical
2940,2900,2500,
1480, 1460, 1380, 1200, 1050, 1015,
UV h max EtoH nm (6): 220 (sh. 1l,lOO), 271 (2020), 279 (1980). EIMS m/z: (rel.
intensity): 234 ]M’]
(1 l), 203(49), 187( 100). 172(68), 157(49). lH-NMR
(6H, s), 1.32 (3H. s) 1.65 (lH, d, 5=14.0 Hz), 2.05 (lH,d, 5=12.0 Hz), 3.85 (lH, d, &12.0 6.
26, 2097-2100.
properties of this compound are as follows. Colorless amorphous.
IR v max Fi1m cm -l: 3350,3250,2980, 950,835.
1985,
L&t.
The physicochemical 2970,2945,2890,
(TMS, CD3COCD3):
1.20
J=14.0 Hz), 2.38 (3H, s), 3.63 (lH, d,
Hz), 4.70 (2H, s), 7.00 (2H, s).
properties of this compound are as follows. Colorless oil. IR v max Film cm -1: 1705, 1750, 1460, 1380, 1260, 1240, 1210, 1060, 1040,910,810.
UV A
max EtCH nm (4: 271 (2230). 279 (2330). EIMS m/z: (rel. intensity): 388]M+J (lo), 329(14), 315(70), 256(54), 228(12), 199(16), 185(20). lH-NMR
(TMS CDC13): 1.29 (6H, s), 1.52 (3H, s)
1.83 (lH, d, 5=14.0 Hz),2.02 (3H. s), 2.18 (3H, s), 2.27 (3H, s), 2.40 (lH, d, J=14.0 Hz), 2.55 (2H, m), 4.16 (2H, br s), 5.35 (lH, br dd, 5=3.5, 8.0 Hz), 6.23 (lH, br t, 3=8.0 Hz), 7.10 (2H, s). 7.
Horeau, A.; Kagan, H. B. Tad&on,
8.
Okuda, T; Harigaya, S; Kiyomoto,
9.
Frankland, B.; Wareing,P. F. NATURE, 1960,
(Received
in Japan
17 July
1995; revised
1964,
20, 243 l-2441.
A. Chem.Pharm Bull. 1964,
21 August
i2,
304-306.
18.5. 255-256.
1995; accepted
25 August
1995)