bUoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mauz, Nizwa, Oman;
cDepartment of Chemistry, University of Malakand, Chakdara, Dir (L), Pakistan;
dDepartment of Chemistry, CIIT, Abbottabad Campus, Abbottabad 22060, Pakistan;
eDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan;
fDepartment of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan;
gDepartment of Chemistry and Polymer Science, University of Stellenbosch, P/Bag X1, Matieland 7602, South Africa;
hInstitute of Microbiology, University of Braunschweig, Spielmannstra?e 7, Braunschweig 38106, Germany
Since the discovery of the world’s first billion-dollar anticancer drug,paclitexel (Taxol) isolated fromPestalotiopsis microspora,a fungus that colonizes the Himalayan yew tree Taxus wallichiana without causing apparent injury to the host plant,interest has been growing in symptomless parasitic fungi,termed the ‘endophytes’ . Some species of endophytic fungi have been identified as sources of anticancer,antidiabetic,insecticidal and immunosuppressive compounds. Further,endophytic fungi may also produce metabolites having a thermoprotective role. The term endophyte is broadly applied to fungi (or bacteria) which live within plant tissues, for all or part of their life cycle without causing any apparent infections . Continuing our work on the characterization of structurally novel and/or biologically active metabolites isolated from fungal endophyte cultures [2, 3, 4, 5, 6, 7],we investigated the ethyl acetate extract of the endophytic fungiSeimatosporiumsp. and Colletotrichum sp. This investigation led to the isolation and structural determination of two new compounds,named seimatoric acid (1) and colletonoic acid (6),together with six known compounds viz.,2-hydroxymethyl-4b,5a,6b-trihydroxycyclohex-2-enone (2),(+)-phyllostine (3),(+)-epiepoxydon (4) and (+)-epoxydon monoacetate (5). Seimatoric acid (1) was also isolated from another taxonomical unidentified fungal strain 4295 in our group. Details of the isolation,structure elucidation,and biological activity of the new and known compounds are reported herein. 2. Experimental 2.1. General experimental procedures
Column chromatography was performed with commercial silica gel (Merck,0.040-0.063 mm) and Sephadex LH-20 (Amersham Biosciences). Analytical and preparative thin-layer chromatography (TLC) was performed on precoated silica gel plates Merck G60 F-254 or G50 UV-254. Optical rotation was recorded with a Perkin-Elmer 241 MC polarimeter at the sodium D-line. IR spectra were recorded with a Nicolet-510P spectrophotometer. 1H NMR and13C NMR spectra were recorded with a Bruker Avance 500 (500 MHz for 1H and 125 MHz for 13C) spectrometer. MS and HRMS were recorded in the EI mode with MAT 8200 and Micromass LCT mass spectrometers. Microbiological methods and culture conditions are as described previously [8, 9]. 2.2. Culture,extraction,and isolation
The endophytic fungusSeimatosporiumsp. (internal strain no. 8883) was isolated fromSalsola oppositifolia,from Gomera (Spain), and was cultivated on a biomalt solid agar medium (12 L,5% (w/v)) at room temperature for 21 days. The cultures were then extracted with ethyl acetate to afford a residue (3.5 g) which was separated into two fractions by column chromatography on silica gel with a gradient ofn-hexane/ethyl acetate (90:10,50:50,0:100) as the eluent. The least polar fraction 1 (2.1 g) contained mainly fatty acids and lipids. The next polar fraction 2 (1.1 g) was further separated by column chromatography on silica gel withn-hexane/ ethyl acetate (10:1,5:1) to give six sub-fractions (F1-6). The subfractions F1,2were further purified by silica gel column chromatography (CC) and preparative TLC with n-hexane/ethyl acetate (10:1 to 5:1) to give pure compounds 1 (1.5 mg),2(10 mg),3 (3 mg),4(4 mg),and 5 (4.5 mg).
The endophytic fungusColletotrichumsp. from Umbelliferae and from Prerow internal strain 6728 was used for the next isolation. It was cultivated at room temperature for 28 days [8, 9] on biomalt solid agar medium. The culture media were then extracted with ethyl acetate to afford of a residue (3.2 g) which was separated into eight fractions (F1-8) by column chromatography (CC) on silica gel,using gradients ofn-hexane/ethyl acetate (95:5, 80:20,50:50,0:100). The fractions F2,3 were further separated by silica gel column chromatography (CC) and preparative TLC withnhexane/ethyl acetate (9:1,8:3,and 6:4) to give pure colletonoic acid (6,5.5 mg).
Seimatoric acid (1): Yellow solid. IR (KBr,cm-1 ): 3320,2920, 2844,1710,1665,1580,1420,1266,1111,1024,809; UV (MeOH) λmax(loge) 246 (3.39),237 (3.35),194 (0.57). 1H NMR (500 MHz, CDCl3): δ 2.16 (s,3H,3'-Me),2.75 (t,2H,J= 6.5 Hz,H-2),3.32 (t,2H, J= 6.5 Hz,H-3),3.75 (s,3H,4'-OMe),3.87 (s,3H,2'-OMe),6.67 (d, 1H,J= 8.7 Hz,H-5'),7.67 (d,1H,J= 8.7 Hz,H-6'),12.48 (s,1H, CO2H). 13C NMR (125 MHz,CDCl3): δ 8.99 (3'-Me),28.4 (C-2),36.9 (C-3),55.8 (4'-OMe),61.9 (2'-OMe),102.0 (C-5'),120.3 (C-3'),124.8 (C-1'),128.9 (C-6'),159.3 (C-4'),163.3 (C-2'),177.3 (C-1),199.1 (C-4); EIMS: m/z(%) = 252.1 [M+] (12.3),101 (33.4). HREIMS: m/z 252.0990 (Calcd. for C13H16O5 252.0998).
Colletonoic acid (6): White amorphous solid; Mp: 137-139°C; IR (KBr,cm-1 ): 3460 (OH),2854 (CHO),1701 (C==O),1576,1429, (aromatic C==C),1372 (C-O);λmaxnm (log ω): 260 (3.50),245 (4.10),205 (3.50); 1H NMR (500 MHz,CDCl3): δ 2.39 (s,3H,3-CH3), 3.85 (s,3H,5-OMe),6.35 (s,1H,H-6),10.45 (s,1H,CHO),11.50 (s,1H,CO2H),12.45 (s,1H,2-OH); 13C NMR (125 MHz,CDCl3): δ 23.1 (3-Me),53.4 (5-OMe),102.1 (C-4),107.2 (C-1),110.3 (C-6), 135.1 (C-3),159.1 (C-5),162.1 (C-2),179.3 (CO2H),192.2 (CHO); EIMS m/z (rel. int. %): m/z(%) = 210 [M+] (22.2); HREIMS: m/z 210.0520 (Calcd. for C10H10O5 210.0528). 2.3. Agar diffusion test for biological activity
Compound 6 was dissolved in acetone at a concentration of 1 mg/mL. Fifty microliters of the solution (50mg) was pipetted onto a sterile filter disk (Schleicher & Schuell,9 mm),which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organism . The test organisms used were the Gram-positive bacterium,Bacillus fusca(both grown on NB medium),the fungi Microbotryumviolaceum,and the alga Chlorella fusca(all grown on MPY medium). Reference substances were penicillin, nystatin,actidione,and tetracycline. Commencing at the middle of the filter disk,the radius of the zone of inhibition was measured in millimeters. These microorgansims were chosen because (a) they are nonpathogenic and (b) they had in the past proved to be accurate initial test organisms for antibacterial,antifungal,and antialgal/herbicidal activities. 3. Results and discussion 3.1. Structure elucidation
The molecular formula of seimatosporic acid (1) was assigned C13H16O5 on the basis of HREIMS with a molecular peak at m/z 252.0990 and 1H NMR and 13C NMR spectral analyses (see Section 2). The IR spectrum showed absorption for a benzene ring (1580 and 1420 cm-1 ),hydroxyl group (3320 cm-1 ),aliphatic chain (2920 cm-1 ),and two carbonyls (1665 and 1710 cm-1 ). The 1H NMR spectrum of1displayed an AB spin system for two protons of a 1,2,3,4-tetrasubstituted aromatic ring at δ 6.67 (d,1H, J= 8.7 Hz,H-5'),7.67 (d,1H,J= 8.7 Hz,H-6'),and the 13C NMR and DEPT spectra exhibited two CH-signals atd102.0 (C-5') an δ 128.9 (C-6'),and four pure C-signals at δ 120.3 (C-3'),124.8 (C-1'),159.3 (C-4') and 163.3 (C-2') accounted for the 1,2,3,4-tetrasubstituted aromatic ring. The 1H NMR spectrum of1also showed signals for one aromatic methyl group atd2.16 (3'-Me) and two methoxy signals atd3.75 (4'-OMe) and 3.87 (2'-OMe). The relative positions of the two methoxy and one methyl group of the 1,2,3,4-tetrasubstituted aromatic ring were confirmed from the HMBC correlations: 4'-OMe to C-4';2'-OMe to C-2';3'-Me to C-2',C-3', and C-4'(Fig. 1).
|Fig. 1. Structures of compounds 1-6 isolated from Seimatosporium sp. and Colletotrichumsp.|
The 1H NMR spectral data of 1 further confirmed one 4-oxobutanoic acid side chain moiety attached at C-1'in the molecule,as represented by two methylene triplet signals at δ 2.75 (H-2) and 3.32 (H-3) and one carboxyl proton singlet at δ 12.48 (CO2H). The 4-oxobutanoic acid moiety was further confirmed from the 13C NMR spectrum which showed signals for one carboxylic carbonyl δ 177.3,one ketone δ 199.1 and two methylene signals at δ 28.4 (C-2) and 36.9 (C-3). This was further supported by the significant fragment at m/z101 [(C4H5O3]+,in the EIMS as well as COSY and HMBC correlations (Fig. 2).
|Fig. 2. Key COSY and HMBC correlations for seimatosporic acid (1) and colletonoic acid (6).|
The position of attachment of the 4-oxobutanoic acid moiety at C-1' was confirmed from HMBC correlations of H-3 to C-10 and H-6 0 to C-4. Finally the structure of 1 was confirmed from COSY,HMQC, and especially important HMBC correlations which are illustrated in Fig. 2. Consequently,the structure was established to be 4-(2',4'-dimethoxy-3'-methylphenyl)-4-oxobutanoic acid (1),named seimatoric acid,after the producing organism,Seimatosporiumsp.
The molecular formula of colletonoic acid 6 was assigned as C10H10O5 on the basis of HREIMS with a molecular peak at m/z 210.0520 and 1H NMR and 13C NMR spectral analyses (Fig. 2). IR absorption bands at 3460 and 1576 cm-1 indicated the presence of hydroxyl group and double bond,respectively. The 1H NMR spectrum displayed signals for one aromatic proton at δ 6.35 (H-6), a singlet at δ 10.45 (CHO),and one aromatic methyl group at δ 2.39 (3-CH3). This is supported by a signal for the aromatic methine carbon at δ 110.3 (C-6),one aldehyde at δ 192.2 (CHO),and one aromatic methyl group signal at δ 23.1 (3-Me) in the 13C NMR spectrum. The NMR spectra also displayed signals for one methoxy group at δ 3.85 (5-OMe);δC 53.4 (5-OMe),one CO2H group atd 11.50;δC 179.3 (CO2H) and one chelated hydroxyl group at δ 12.45 (2-OH);δC 162.1 (C-2). The chelated hydroxyl proton was placed at C-2,based on its HMBC correlations with C-2 (Fig. 2). Similarly,the aromatic proton at H-6 from its HMBC correlations with C-1,C-2, C-4,and C-5. Positions of the methoxy,CHO,CO2H,and methyl groups were confirmed from the HMBC correlations: OMe to C-5; CO2H to C-4; Me to C-2,C-3,and C-4. Consequently,the structure was established to be 4-formyl-3-hydroxy-6-methoxy-2-methylbenzoic acid 6,named colletonoic acid,after the producing organism,Colletotrichumsp.
Known compounds 2-hydroxymethyl-4β,5α,6β-trihydroxycyclohex-2-enone (2) ,(+)-phyllostine (3) ,(+)-epiepoxydon
(4) ,and (+)-epoxydon monoacetate (5)  were identified by
comparison with published data.
3.2. Biological activity
The antibacterial,fungicidal,and algicidal properties of
colletonoic acid (6) is compiled in Table 1 in comparison to a
number of standard antibiotics and the solvent acetone. Colletonoic acid (6) was tested in an agar diffusion assay for its antifungal,
antibacterial,and algicidal properties toward Microbotryum
violaceum,B. fusca,and C. fusca(Table 1). Colletonoic acid
(6) displayed good antifungal,antibacterial,and algicidal properties toward M. violaceum,B. fusca,and C. fusca. Assuming
that the metabolites produced in culture are also synthesized in
plants,they could,for example,play a role in inhibiting
competitive microorganisms within the endophyte’s natural
habitat. Additionally,due to the fact that a broad range of
microorganisms are inhibited,it would also be interesting to learn
whether colletonoic acid (6) is generally cytotoxic. Unfortunately,
the amount of compound 1 was not enough to test its activity and
antimicrobial activities of compounds 2-5 has already been
reported in our group .
We thank BASF AG and the BMBF (Bundesministerium fu¨r
Bildung und Forschung,No. 03F0360A).
The antibacterial,fungicidal,and algicidal properties of colletonoic acid (6) is compiled in Table 1 in comparison to a number of standard antibiotics and the solvent acetone. Colletonoic acid (6) was tested in an agar diffusion assay for its antifungal, antibacterial,and algicidal properties toward Microbotryum violaceum,B. fusca,and C. fusca(Table 1). Colletonoic acid (6) displayed good antifungal,antibacterial,and algicidal properties toward M. violaceum,B. fusca,and C. fusca. Assuming that the metabolites produced in culture are also synthesized in plants,they could,for example,play a role in inhibiting competitive microorganisms within the endophyte’s natural habitat. Additionally,due to the fact that a broad range of microorganisms are inhibited,it would also be interesting to learn whether colletonoic acid (6) is generally cytotoxic. Unfortunately, the amount of compound 1 was not enough to test its activity and antimicrobial activities of compounds 2-5 has already been reported in our group .
We thank BASF AG and the BMBF (Bundesministerium fu¨r Bildung und Forschung,No. 03F0360A).
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