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Metabolites from static cultures of the fungus Xylaria badia.

Introduction

Xylaria badia is found worldwide (1). The isolate that was examined for its secondary metabolites was collected from Thailand. The culture medium of X. badia produced two compounds. 8-methyl-3, 4-dihydro-1H-isochromen-1-one and 2-(hydroxymethyl)-6-[(8-hydroxy-1-naphthyl)oxy] tetrahydro-2H-pyran-3,4,5-triol. The mycelium contained only fatty material.

Results and discussions

Characterisation of ((R)-Mellein)

The dark brown gum (5.0 g) of crude extract from culture medium of X. badia was chromatographed over silica gel in a column of size 80 cm x 2.5 cm. The column was eluted with toluene, ethyl acetate and acetic acid (50:49:1) and the eluent collected in volumes of 3.0 ml.

Tubes (55-74), fraction 3 gave a yellowish oil (150 mg). The oil was triturated with n-hexane to give a white powdery solid (12 mg). The solid was recrystallised from the same solvent system yielding white crystals (8 mg). mp 48-49 [degrees]C, m/z 178 ([M.sup.+]), ([C.sub.10][H.sub.10][O.sub.3]). [[[alpha]].sup.23.sub.D]-92[degrees] (c = 0.93, MeOH), IR (KBr/cm) 3620-3300, 1670, 1615 and 1580. [sup.1]H NMR determined in CD[Cl.sub.3] had a strong singlet at [[delta].sub.H] 11.02 indicating the presence of a chelated OH. Three signals were observed in the aromatic region of this spectrum ([[delta].sub.H] 6.50-8.5), two doublets at [[delta].sub.H] 6.69 (J 7.39 Hz) and [[delta].sub.H] 6.89 (J 8.4 Hz) coupled to a pseudo triplet at 7.40 (J 7.6 Hz).

The coupling constants are typical ortho coupling constants. In the [sup.13]C NMR determined in CD[Cl.sub.3, signals for six aromatic carbons occur at [[delta].sub.C] 108.36, 116.31, 117.99, 136.24, 139.47 and 162.25. Three of these aromatic protons are quaternary protons, suggesting that the aromatic portion of this compound is tri-substituted as shown in fragment (A). The phenolic nature of the compound was confirmed by treating a solution of the compound with ferric chloride to give violet colouration. The molecular formula of the compound was deduced to be [C.sub.10][H.sub.10][O.sub.3].

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Thus, [C.sub.6][H.sub.4]O had been accounted for, leaving [C.sub.4][H.sub.6][O.sub.2]. In the high field region of the [sup.1]H NMR there are three signals at [[delta].sub.H] 4.70 (1H, m), [[delta].sub.H] 2.91 (2H, d, J 7.1 Hz) and [[delta].sub.H] 1.53 (3H, d, J 6.4 Hz). The position of the methine proton at [[delta].sub.H] 4.70 indicates that it is attached to a C-O group. A [sup.1]H-[sup.1]H COSY spectrum showed the presence of subunit (B). A methyl carbon at [[delta].sub.C] 20.85, a methylene carbon at [[delta].sub.C] 34.68 and a methine at [[delta].sub.C] 76.79 in the [sup.13]C NMR spectra support the above proposal. One carbon and an oxygen atom are left unaccounted for. On TLC, the compound gave a yellow colour with 2,4-DNP spray reagent, indicating the presence of a C=O group. In the [sup.13]C NMR spectrum, there is a quaternary carbon at [[delta].sub.C] 170.06, typical of a carbonyl of a lactone. The phenolic fragment (A) might be joined to fragment (C).

The orientation of fragment (A) and (C) is revealed by the chelation of the phenolic OH with the carbonyl group. The chelated OH occurs at [[delta].sub.H] 11.02, indicating that the OH is in the neighbourhood of the carbonyl group. The optical rotation is negative. This gave the compound as R-(-)-mellein (1).

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A summary of the [sup.1]H and [sup.13]C NMR assignments is shown in Table1

Mellein is a common secondary metabolite found in both higher and lower plants. It was first isolated in 1933 from Aspergillus melleus (2). Later researchers worked out the stereochemistry of mellein. It was found to exist naturally in two main forms; the more common form R-(-)-mellein (1) and the less common isomer S-(+)-mellein (2)

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Table 2 is a summary of the source and physical properties of R-(-)-mellein reported in literature

Characterisation of Naphthol glucoside

Tubes (215-304) fraction 6, was brownish oil. The oil (1.53 g) was triturated with ethyl acetate and left for 24 hrs. A brown solid was obtained. This was filtered off and dried under reduced pressure to give a light brown solid (514 mg). The solid was recrystallised from ethyl acetate yielding light brown plates (480 mg). The compound was insoluble in light petroleum, acetone and water. It was however soluble in methanol. Its [R.sub.f] values were very low in the common eluents such as toluene, ethyl acetate and acetic acid (50:49:1) [R.sub.f] (0.15), and chloroform methanol (95:5). [R.sub.f] (0.21). The molecular formula was found to be [C.sub.16][H.sub.18][O.sub.7], mp. 216-218 [degrees]C, [[[alpha]].sup.23.sub.D] + 203.8. (c = 0.85, MeOH), m/z 322 ([M.sup.+]), IR (KBr/cm) 3605, 3005, 2890, 1600 and 1450. TLC plates of the compound gave a brown colour with diazotised p-nitroaniline spray reagent. The compound reacts with alcoholic solution of ferric chloride to give a violet colour, indicating that it is phenolic. Supporting evidence is found in the IR where a peak occurs at 3605 [cm.sup.-1] The [sup.1]H NMR spectrum determined in CD[Cl.sub.3] showed a low field group of protons ([[delta].sub.H] 6.78-7.46) and a high field group of protons ([[delta].sub.H] 3.46-3.86). The latter group is suggestive of glycoside protons.

Subunit A

In the low field region of the [sup.1]H NMR spectrum three distinct group of signals occur at [[delta].sub.H] 7.46 (2H, m), 7.27 (3H, m), 6.77 (1H, dd, J 5.9, 3.0 Hz). Ten aromatic carbons are observed in the [sup.13]C NMR. Four of these are quaternary and the rest are aromatic methine carbons. Two of the quaternary carbons are attached to oxygen ([[delta].sub.C] 153.73 and 154.54). These features suggested a substituted naphthalene diol. The [sup.1]H-[sup.1]H DQF COSY spectrum shows coupling amongst the three groups of protons. Two possible structures A1 and A2 could be drawn to represent the above spectra data.

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ACDLabs software was used to calculate the chemical shifts of the carbon atoms in these two fragments. The experimental carbon-13 chemical shifts were compared to the calculated chemical shifts. (Table 4.5). The variable (X) was replaced with a methoxy group in order to obtain the calculated values.

The fragment A1 compared more favourably with the experimental results. In the [sup.1]H-[sup.1]H COSY spectrum, there is a strong coupling between the proton at [[delta].sub.H] 6.77 and the protons at [[delta].sub.H] 7.27. The latter protons are in turn coupled to the group of protons at [[delta].sub.H] 7.46. The integration of these protons at resonance positions [[delta].sub.H] 7.46, 7.27 and 6.77 is in the ratio 2:3:1. The likely arrangements of the protons on A1 is as shown in A3.

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Subunit B

The protons at high field in the [sup.1]HNMR spectrum are within the range [[delta].sub.H] 3.45-3.86. This suggests that the protons might be attached to C-O groups. The integration indicates the presence of six protons. [sup.13]C measurements revealed that the six protons are due to four methines and a methylene. The molecular formula of the compound was found to be [C.sub.16][H.sub.18][O.sub.7]. Subunit A accounted for [C.sub.10][H.sub.7]O, leaving C6H11O6. On the basis of this molecular formula and the evidence above, the second subunit is most likely a glucose fragment.(B).

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In the [sup.1]H-[sup.1]H COSY spectrum, the proton at [[delta].sub.H] 3.45 is coupled to protons at [[delta].sub.H] 3.73 and 3.82. The HMBC spectrum (figure 4.21), shows correlation between the proton at 3.45 and carbon atoms at [[delta].sub.C] 61.15 and 71.54. This high field proton is attached to the methine carbon [[delta].sub.C] 69.68 according to the HMQC. In the latter spectrum, the carbon at [[delta].sub.C] 61.15 has two proton correlations indicating that it is a methylene carbon. A possible fragment in subunit B is (B1).

Fragment B1 accounts for [C.sub.4][H.sub.8][O.sub.3], leaving [C.sub.2][H.sub.3][O.sub.3]. A methine proton down field at [[delta].sub.H] 5.57 suggests a proton on a carbon attached to two oxygen atoms or an [sp.sup.2] hybridised carbon atom. In the [sup.1]H-[sup.1]H COSY spectrum, this proton is coupled to the proton at [[delta].sub.H] 3.69 which appears as a doublet of a doublets (J 9.4, 3.9, Hz), because it is in turn coupled to the proton at [[delta].sub.H] 3.82. The HMBC spectrum shows a two and a three bond correlation between the low field methine proton at [[delta].sub.H] 5.57 and carbon atoms at [[delta].sub.C] 71.54 and 74.09. Subunit B might have its other section as shown in (B2).

Combining fragments B1 and B2 gives Subunit (B). Subunit (A) and (B) are linked to give the Naphthol glucoside (2). Evidence of the link is given by the three bond correlation between the methine proton at [[delta].sub.H] 5.57 and the aromatic carbon at [[delta].sub.C] 153.73 present in the HMBC.

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Experimental

Isolation of (R)-mellein

The crude extract (5.0 g) from a second culture of X. badia was chromatographed over silica gel in a column (80 x 2.5 cm) and eluted with toluene, ethyl acetate and acetic acid (50:49:1). Fraction 3 (tubes 55-74) gave a yellowish oil (150 mg). n-hexane (1.0 ml) was added to the oil and warmed to boiling point. The solution was filtered hot and allowed to cool overnight. A white solid (12 mg) was obtained. The solid was recrystallised in the same solvent system to give R-(-)-mellein white crystals (8 mg), [C.sub.10][H.sub.10][O.sub.3]; mp. 48-50 [degrees]C, (lit.7, 47[degrees]C), m/z 166 ([M.sup.+]), [[[alpha]].sup.23.sub.D] -92[degrees] [c=0.93, MeOH], [v.sub.max] (KBr) [cm.sup.-1] 3620-3300, 1670, 1615 and 1580. [[delta].sub.H] (CD[Cl.sub.3]) 1.53 (11-C[H.sub.3], d, J 6.4 Hz), 2.91 (4-C[H.sub.2], d, J 7.1 Hz), 4.70 (3-CH, m). 6.69 (Ar-CH, d, J 7.4 Hz), 6.88 (Ar-CH, d, J 8.4 Hz), 7.40 (Ar-CH, t, J 7.3, Hz). [[delta].sub.H] (CDCb) 20.85 (11-C[H.sub.3]), 34.68 (4-C[H.sub.2]), 76.79 (3-CH), 108.36 (7-C), 116.31 (5-CH), 117.99 (6-CH), 136.24 (9-CH), 139.47 (10-C), 162.25 (8-C) and 170.06 (1-C=O).

Purification of Naphthol glucoside

Fraction 7 (tubes 215-304) from the column chromatography that gave R-(-)-mellein and coriloxin afforded a brownish liquid (1.53 g) when the solvent was removed at low pressure on the rotary evaporator. The oil was triturated with ethyl acetate (3 ml) and left at room temperature for 24 h. A brown solid precipitated from the solution. The solid (514 mg) was recrystallised from ethyl acetate to give Naphthol glucoside as light brown plates (480 mg), molecular formula found ([C.sub.16][H.sub.18][O.sub.7]), mp 216-218 [degrees]C (lit.4, 217 [degrees]C) m/z 222 [M.sup.+], [v.sub.max] (KBr) [cm.sup.-1]; 3605, 3005, 2890, 1600, and 1450. [[delta].sub.H] (C[D.sub.3]OD) 3.45 (1H, t, J 9.2 Hz), 3.69 (1H, m) 3.73 (1H, m), 3.82 (1H, t, J 9.2 Hz), 3.86 (1H, m), 5.57 (1H, d, J 3.8 Hz), 6.77 (1H, dd, J 5.9, 3.0 Hz), 7.27 (3H), m) and 7.5 (2H, m). [[delta].sub.C] (C[D.sub.3]OD) 61.15 ([7.sup./]-C[H.sub.2]), 69.68 ([3.sup./]-CH), 71.54 ([6.sup./]-CH), 73.57 ([2.sup./]-7CH), 74.09 ([4.sup./]-CH), 101.13 ([6.sup./]-CH), 110.49 (7-CH), 110.49 (4-CH), 115.76 (9-C), 118.78 (2-CH), 122.93 (5-CH), 125.69 (3-CH), 127.00 (6-CH), 137.01 (10-C), 153.73 8-C) and 154.54 (1-C).

Conclusion

Mellein is a common metabolite found in most lower and higher plants. The R-mellein isolated from X. badia is very similar in physical properties to that reported from Lasiodiplodia spp (6). Naphthol glucoside is a known compound. It was first isolated from cultures of Sclerotinia sclerotiorum by Starratt et al (8). The group also showed the effect of tricyclazole on its production. Cameron et al (9) had however, synthesised this compound as far back as 1965. They showed that the anumeric proton for [alpha]-naphthol glucoside has a chemical shift of [[delta].sub.H] 5.41 and [beta]-naphthol glucoside [[delta].sub.H] 5.18. This suggests that the isolated compound is most likely [alpha]-naphthol glucoside. This is however the first report of the isolation of these compounds from the fungus Xylaria badia.

Reference

[1] E. Nishakawa; J. Agri. Chem. Soc., Japan, 1933, 9, 772.

[2] M. J. Garson and J. Staunton, J. Chem. Soc. Perkin Trans. 1 1984, 1021.

[3] M. Sasaki, Y. Kaneko, K. Oshita, H. Takamatsu and Y. Asao, J. Agr. Biol. Chem., 1970, 34, 1296.

[4] J. R. Anderson, R. L. Edwards and A. J. S. Whalley, J. Chem. Soc. Perkin Trans. 1, 1983, 2185.

[5] K. Mori and A. K. Gupta, Tetrahedron, 1985, 41, 5295.

[6] M. J. Garson and J. Staunton, J. Chem. Soc. Perkin Trans. 1, 1999, 715.

[7] C. Dimitriadis, M. Gill and M. F. Harte, Tetrahedron-Asymmetry, 1997, 8, 2153.

[8] A. N. Starratt, L. M. Ross, and G. Larovits, J. Microbiol. 2002, 48, 320-325

[9] D. W. Cameron, H. W. S. Chan and D. G. I. Kingston, J. Chem. Soc., 1965, 4363-4368.

E.K Oppong (1), R.L. Edwards (2), D.J. Maitland (3) and R. Hanson (4)

(1,4) Dept. of Science Education, University of Education, P.O. Box 25, Winneba, Ghana

(2,3) Dept. of Chemistry and Forensic Science, University of Bradford, Richmond Road, West Yorkshire, BD7 1DP, United Kingdom
Table 1: [sup.1]H and [sup.13]C NMR designation of
R-(-)-mellein.

([[delta].sub.H]) ([[delta].sub.C])

1.53 (3H,d, J 6.3 Hz) 20.85 34.68
2.91 (2H, d, 7.1 Hz) 76.79 116.31
4.70 (1H, m) 117.99 136.24
6.69 (1H, d, J 7.4 Hz) -- 108.36
6.88 (1H, d, J 8.4 Hz) 139.47 162.25
7.40 (1H, d, J 7.6,) 11.03 170.06
(1H, s(br))

([[delta].sub.H]) [C.sub.#]

1.53 (3H,d, J 6.3 Hz) 1 4
2.91 (2H, d, 7.1 Hz) 3 7
4.70 (1H, m) 5 6
6.69 (1H, d, J 7.4 Hz) -- 9
6.88 (1H, d, J 8.4 Hz) 10 8
7.40 (1H, d, J 7.6,) 11.03 1
(1H, s(br))

Table 2: Source and physical properties of R-(-)-mellein reported
in literature.

Melting point Optical rotation Source
([degrees]C)

56 -102.5[degrees] (c 1.0, Aspergillus
 CH[Cl.sub.3] at 25[degrees]C) onika (3)
58 -100[degrees] (c 1.0, Hypoxylon
 CH[Cl.sub.3] at 22[degrees]C) howieanum (4)
56-58 -80[degrees] (c 0.02, Aspagillus
 CH[Cl.sub.3] at 22[degrees]C) melleus
55-56 -100.8[degrees] (c 1.01, Synthesis (5)
 CH[Cl.sub.3] at 22[degrees]C)
47-48 -94[degrees] (c 0.48, Lasiodiplodia
 CH[Cl.sub.3] at 22[degrees]C) spp. (6)
55.5-56 -101.3[degrees] (c 0.07, Synthesis (7)
 CH[Cl.sub.3] at 26[degrees]C)

13 Table 3: ACDLabs [sup.13]C NMR of A1 and A2 compared with
experimental values.

A1 ([[delta]. A2([[delta]. ([[delta].sub.C]) [C.sub.#]
sub.C] cal.) sub.C] cal.)

155.24 153.54 156.62 151.81 154.54 153.73 1 8
135.29 126.96 125.41 123.44 137.01 127.00 9 6
126 96 126.72 126.63 124.66 125.69 122.93 3 5
120.86 113.28 115.05 114.28 118.78 115.76 4 7
113.41 110.36 106.61 110.49 110 49 10 2
106.08

Table 4: [sup.1]H and [sup.13]C NMR assignments of naphthol
glucoside.

([[delta].sub.H]) ([[delta].sub.C]) Assignment [C.sub.#]

3.45 (1H, t, J 69.68 CHOH [3.sup./]
 9.2 Hz)
3.69 (1H, dd, J 71.54 CHOH [5.sup./]
 3.8, 9.4 Hz)
3.73 (1H, m) 61.15 HCHOH [7.sup./]
3.73 (1H, m) 73.57 OCH [2.sup./]
3.82 (1H, t, J 74.09 CHOH [4.sup./]
 9.2 Hz)
3.86 (1H, m) 61.15 HCHOH [7.sup./]
5.57 (1H, d, J 101.13 OCHO [6.sup./]
 3.8 Hz)
6.77 (1H, dd, J 110.49 Ar-CH 7
 5.9, 3.0 Hz)
7.27 (3(1H), m) 118.78,125.69, 3Ar-CH 2,3,6
 127.00
7.46 (2Qh), m) 110.49,122.93, 2Ar-CH 4,5
- 154.54 Ar-CO 1
 153.73 Ar-COH 8
 115.76 Ar-C 9
 137.01 Ar-C 10
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Author:Oppong, E.K; Edwards, R.L.; Maitland, D.J.; Hanson, R.
Publication:International Journal of Applied Chemistry
Date:Sep 1, 2010
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