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Bromophenols Identified in a Marine Ascomycete and its Culture Medium
Institute of Physiological Botany, University of Uppsala, Sweden
Bromophenols Identified in a Marine Ascomycete and its Culture Medium MARIANNE PEDERSEN and NILS FRIES Received November 12, 1976 . Accepted December 1, 1976
Summary Four bromo-compounds have been detected in an axenically cultured marine Ascomycete isolated from the brown alga Ascophyllum nodosum. The fungus was grown in a synthetic medium supplemented with 100 mg .1- 1 KBr, in a culture shaken for 180 days. One of the bromocompounds was identified as 2,3-dibromo-4,5-dihydroxybenzyl alcohol. The culture filtrate did not contain this compound but another brominated phenol, 3,5-dibromo-phydroxybenzyl alcohol, and the other three bromo-compounds that were present in the Ascomycete.
Key words: marine ascomycete, Ascophyllum nodosum, bromophenols.
Introduction Bromo-compounds have been identified in various marine organisms (SIUDA and DEB ERN ARDIS, 1973). The bromophenols, 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol) and 3,5-dibromo-p-hydroxybenzyl alcohol have previously only been identified in marine algae (for review see WEINSTEIN et aI., 1975), in sea water (PEDERSEN et aI., 1974) and in the culture filtrate of a blue-green alga PEDERSEN and DASILVA, 1973). They also constitute the two main bromophenols of the red pigment floridorubin isolated from the red alga Lenormandia prolifera (SAENGER et aI., 1976). We now describe the isolation and identification of lanosol from a marine Ascomycete and 3,5-dibromo-p-hydroxybenzyl alcohol from its culture medium.
Material and Methods The marine Ascomycete was isolated from the brown alga Ascophyllum Le Jolis collected in October 1972 at the vicinity of Kristineberg's Biological Swedish West Coast. The mycelium lives as an endophyte in the alga and grew out slowly on medium from pieces of the algal thallus, which had been surface sterilized
nodosum (L.) station at the nutrient agar by treatment
Abbreviations: GC-MS, gas-liquid chromatography - mass spectrometry; TMSi, trimethylsilyl-; DMCS, dimethyldichlorosilane.
z.
Pjlanzenphysiol. Bd. 82. S. 363-366. 1977.
364
M. PEDERSEN and N. FRIES
with ethyl alcohol (70010) and calcium hypochlorite (cone. 501n.). Since it remained growing as an undifferentiated vegetative, white mycelium its taxonomic position could not be established with certainty. However, electron microscopy showed that its hyphal walls and septal pores were of the Ascomycete type. Its omnipresence as an endophyte in Ascophyllum [and Pelvetia canaliculata (L.) DEC. et THUR.] but absence from other brown algae investigated, indicated that it could be identical with Mycosphaerella ascophylli Cotton (WEBER, 1967; KOHLMEYER, 1972). The Ascomycete was axenically grown in a liquid medium of the following composition: glucose 20 g, KNO a 7.2 g, K 2 HP0 4 1.2 g, MgS0 4 • 7 H 20 0.5 g, KBr 0.1 g, mixture of 8 vitamins, trace elements (Ca2+, Zn 2+, Mn2+, Fe2+), and distilled water 1 1. The culture flasks (300 ml Erlenmeyer) each contained 100 ml medium and were shaken at 20 DC for 180 days. The mycelia from five cultures were collected, washed with distilled water, deep-frozen, pulverized with liquid N2 and extracted with 80010 Me2CO for 20 min in an ultrasonic bath (Branson Instr. Co., Stamford, Conn., USA) under cooling to 30 DC. The Me2CO was removed after filtration by rotary evaporation. Removal of possibly occurring esterified sulfates was achieved by acidification to pH 2 with 1 N HCl and simultaneous heating for 10 min. On cooling, the resulting solution was extracted 3 times with 30 ml aliquots of EtOAc; the fractions thus obtained were pooled and the solvent was evaporated. The residue was redissolved in a small volume of EtOAc, transferred to a microsilylation vessel and concentrated to dryness under nitrogen and further over phosphorus pentoxide to ensure the absence of any traces of water. BSA [bis (trimethylsilyl) acetamide] and MeCN were added at 20,ul each, the entire mixture heated at 60 DC for 15 min and subjected to GC-MS. The culture filtrate (130 ml) was acidified to pH 2, heated to 70 DC for 10 min, extracted 3 times with 100 ml EtOAc, and then subjected to the same extraction and silylation procedure as above. Analyses of the TMSi derivatives were carried out on a FID instrument using a 1.2 m X 4 mm silanized glass column packed with 3 Ofo (w/w) SE 30 on 80/100 mesh acidwashed, DMCS-treated Chromosorb W. A carrier gas (97010 N2 and 3010 H 2 ) flow rate of 50 mllmin was maintained. Injector and detector temperatures were maintained at 220°C and 240 DC, respectively. The mass spectra were determined with a LKB 9000 mass spectrometer coupled to a gas chromatograph described by BERGSTROM (1973). Helium was employed as the carrier gas at the flow rate of 25 mllmin.
Results
MS analyses showed the presence of 2,3-dibromo-4,5-dihydroxybenzyl alcohol in the marine Ascomycete and 3,5-dibromo-p-hydroxybenzyl alcohol in its culture medium together with three unidentified bromo-compounds, which were present both in the Ascomycete and its culture medium. The data for the different compounds are presented in Table 1. The mass spectra for the two bromophenols mentioned, III and V, were in entire agreement with those for authentic samples of them and for the same bromophenols isolated from red algae. Bromo-compound I contains 3 Br atoms and after silylation has a MW of 400. According to the fragmentation pattern of the mass spectra of bromo-compounds II and IV they are the same compound although with 1 and 2 Br atoms, respectively. The loss of two 0- TMSi-groups (M+-89-89) is clearly seen from their mass spectra. Their structures however remain to be elucidated. The dibromo-monohydroxy- and dibromo-dihydroxy-alcohols are known Z. PJlanzenphysiol. Ed. 82. S. 363-366. 1977.
Bromophenols in a marine Ascomycete
365
Table 1: Bromo-compounds from the marine Ascomycete and its culture filtrate. MW (Silylared)
No. of Br atoms
Ascomycete I 400 II 476 III 512 IV 552
3 1 2 2
2 or more 3 2 or more
175 0 210° 215° 220°
Culture medium I 400 V 424 II 476 IV 552
3 2 1 2
2 2 or more 2 or more
175° 203° 210° 220°
No. of TMSi groups
GLC Temp. programmed from 100 0 at 6°/min
Proposed terminal group
CH 20H
CH20H
to be esterified with sulphate at positions 1 and 4 (HODGKIN et aI., 1966; GLOMBITZA and STOFFELEN, 1972; WEINSTEIN et aI., 1975). In the present investigation no attempt has been made to establish whether the bromophenols of the marine Ascomycete occur as benzyl sulphonates. A recent investigation of the red alga Rytiphlea tinctoria (CHEVOLOT-MAGUEUR et aI., 1976) revealed lanosol as being a part of a 5,6,3',5' -tetrabromo-3,4,2',4',6-pentahydroxydiphenylmethane, which also appeared in the red pigment floridorubin as a photosynthetic pigment replacing phycobilins in this alga.
If lanosol constitutes one of the phenols of a photosynthetic pigment its appearance in a marine Ascomycete may be of interest from the point of view of the phylogeny of the Ascomycetes. Some taxonomists [d. MARTIN (1968) for references] have suggested that the Ascomycetes originate from marine red algae. Lanosol has also been identified in the two brown algae Fucus vesiculosus and Ascophyllum nodosum in very small amounts (PEDERSEN and FRIES, 1974; PEDERSEN, 1976). The possibility that marine Ascomycetes living as epi- or endophytes in these brown algae are producing these bromophenols cannot be neglected. No bromophenols have hitherto been identified in other brown or in green algae (PEDERSEN, 1976). In red algae the bromo phenols are only found in Florideae. Members of the family Rhodomelaceae are especially rich in these compounds, while they are lacking in all species belonging to the Bangioideae (PEDERSEN et aI., 1974). The reported presence of lanosol in the water of the Polysiphonia Brodiaei zone (PEDERSEN et a!., 1974), the findings of lanosol and tribromophenols in the culture filtrate of Calothrix brevissima (PEDERSEN and DASILVA, 1973) and now the four bromo-compounds in the culture filtrate of the Ascomycete indicate exudation of
z.
PJlanzenphysiol. Bd. 82. S. 363-366. 1977.
366
M. PEDERSEN and N. FRIES
these compounds surroundings.
from
the
algae
and
the
Ascomycete
into
the
immediate
Acknowledgements We thank Dr. L. FRIES for valuable advice, and Dr. A. VON HOFSTEN for having performed the electron microscopy. The investigation was supported by a grant from the Swedish Natural Science Research Council to Dr. L. FRIES.
References BERGSTROM, G.: Studies on natural odoriferus compounds. VI. Use of a pre-column tube for the quantitative isolation of natural, volatile compounds for gas chromatography / mass spectrometry. Chern. Scr. 4,135-138 (1973). CHEVOLOT-MAGUEUR, A.-M., A. CAVE, P. POTIER, J. TESTE, A. CHIARONI, and C. RICHE: Composes bromes de Rytiphlea tinctoria (Rhodophyceae). Phytochemistry (Oxf.) 15, 767-771 (1976). GLOMBITZA, K.-W., and H. STOFFELEN: 2,3-dibrom-5-hydroxybenzyl-l ',4-, disulfat (Dikaliumsalz) aus Rhodomelaceen. Plant a Med. 22, 391-396 (1972). HODGKIN, J. H., J. S. CRAIGIE, and A. G. McINNES: The occurrence of 2,3-dibromo-benzyl alcohol 4,5-disulfate, dipotassiumsalt in Polysiphonia lanosa. Can. J. Chern. 44, 74-78 (1966). KOHLMEYER, J. & E.: Is Ascophyllum lichenized? Bot. Mar. 15, 109-112 (1972). MARTIN, G. W.: The origin and status of fungi (with a note on the fossil record). In: G. C. AINSWORTH and A. S. SUSSMAN (Eds.), The fungi, Vol. III, pp. 635-648. Academic Press, New York and London, 1968. PEDERSEN, M.: Studies on marine algae with emphasis on some organic compounds produced by these algae. Acta Univ. Ups. Abstr. Upps. Diss. Sci. 355, 1-17 (1976). PEDERSEN, M., and E. J. DASILVA: Simple brominated phenols in the bluegreen alga Calothrix brevissima WEST. Planta (Berl.) 115, 83-86 (1973). PEDERSEN, M., and L. FRIES: Brominated phenols in Fucus vesiculosus. Z. Pflanzenphysiol. 74, 272-274 (1975). PEDERSEN, M., P. SAENGER, and L. FRIES: Simple brominated phenols in red algae. Phytochemistry (Oxf) 13, 2273-2279 (1974). SAENGER, P ., M. PEDERSEN, and K. S. ROWAN: Bromo-compounds of the red alga Lenormandia prolifera. Phytochemistry (Oxf) in press (1976). WEBBER, F. c.: Observations on the structure, life history and biology of Mycosphaerella ascophylli. Trans. Br. Mycol. Soc., 50, 583-601 (1967). WEINSTEIN, B., L. TRACY, R. CLAYTON, E. HARRELL, Jr., M. W. BURNS III, and J. R. WAALAND: Reexamination of the bromophenols in the red alga Rhodomela larix. Phytochemistry (Oxf) 14, 2667-2670 (1975).
MARIANNE PEDERSEN and NILS FRIES, Institute of Physiological Botany, University of Uppsala, S-751 21 Uppsala, Sweden.
Z. Pflanzenphysiol. Bd. 82. S. 363-366. 1977.
Bromophenols Identified in a Marine Ascomycete and its Culture Medium MARIANNE PEDERSEN and NILS FRIES Received November 12, 1976 . Accepted December 1, 1976
Summary Four bromo-compounds have been detected in an axenically cultured marine Ascomycete isolated from the brown alga Ascophyllum nodosum. The fungus was grown in a synthetic medium supplemented with 100 mg .1- 1 KBr, in a culture shaken for 180 days. One of the bromocompounds was identified as 2,3-dibromo-4,5-dihydroxybenzyl alcohol. The culture filtrate did not contain this compound but another brominated phenol, 3,5-dibromo-phydroxybenzyl alcohol, and the other three bromo-compounds that were present in the Ascomycete.
Key words: marine ascomycete, Ascophyllum nodosum, bromophenols.
Introduction Bromo-compounds have been identified in various marine organisms (SIUDA and DEB ERN ARDIS, 1973). The bromophenols, 2,3-dibromo-4,5-dihydroxybenzyl alcohol (lanosol) and 3,5-dibromo-p-hydroxybenzyl alcohol have previously only been identified in marine algae (for review see WEINSTEIN et aI., 1975), in sea water (PEDERSEN et aI., 1974) and in the culture filtrate of a blue-green alga PEDERSEN and DASILVA, 1973). They also constitute the two main bromophenols of the red pigment floridorubin isolated from the red alga Lenormandia prolifera (SAENGER et aI., 1976). We now describe the isolation and identification of lanosol from a marine Ascomycete and 3,5-dibromo-p-hydroxybenzyl alcohol from its culture medium.
Material and Methods The marine Ascomycete was isolated from the brown alga Ascophyllum Le Jolis collected in October 1972 at the vicinity of Kristineberg's Biological Swedish West Coast. The mycelium lives as an endophyte in the alga and grew out slowly on medium from pieces of the algal thallus, which had been surface sterilized
nodosum (L.) station at the nutrient agar by treatment
Abbreviations: GC-MS, gas-liquid chromatography - mass spectrometry; TMSi, trimethylsilyl-; DMCS, dimethyldichlorosilane.
z.
Pjlanzenphysiol. Bd. 82. S. 363-366. 1977.
364
M. PEDERSEN and N. FRIES
with ethyl alcohol (70010) and calcium hypochlorite (cone. 501n.). Since it remained growing as an undifferentiated vegetative, white mycelium its taxonomic position could not be established with certainty. However, electron microscopy showed that its hyphal walls and septal pores were of the Ascomycete type. Its omnipresence as an endophyte in Ascophyllum [and Pelvetia canaliculata (L.) DEC. et THUR.] but absence from other brown algae investigated, indicated that it could be identical with Mycosphaerella ascophylli Cotton (WEBER, 1967; KOHLMEYER, 1972). The Ascomycete was axenically grown in a liquid medium of the following composition: glucose 20 g, KNO a 7.2 g, K 2 HP0 4 1.2 g, MgS0 4 • 7 H 20 0.5 g, KBr 0.1 g, mixture of 8 vitamins, trace elements (Ca2+, Zn 2+, Mn2+, Fe2+), and distilled water 1 1. The culture flasks (300 ml Erlenmeyer) each contained 100 ml medium and were shaken at 20 DC for 180 days. The mycelia from five cultures were collected, washed with distilled water, deep-frozen, pulverized with liquid N2 and extracted with 80010 Me2CO for 20 min in an ultrasonic bath (Branson Instr. Co., Stamford, Conn., USA) under cooling to 30 DC. The Me2CO was removed after filtration by rotary evaporation. Removal of possibly occurring esterified sulfates was achieved by acidification to pH 2 with 1 N HCl and simultaneous heating for 10 min. On cooling, the resulting solution was extracted 3 times with 30 ml aliquots of EtOAc; the fractions thus obtained were pooled and the solvent was evaporated. The residue was redissolved in a small volume of EtOAc, transferred to a microsilylation vessel and concentrated to dryness under nitrogen and further over phosphorus pentoxide to ensure the absence of any traces of water. BSA [bis (trimethylsilyl) acetamide] and MeCN were added at 20,ul each, the entire mixture heated at 60 DC for 15 min and subjected to GC-MS. The culture filtrate (130 ml) was acidified to pH 2, heated to 70 DC for 10 min, extracted 3 times with 100 ml EtOAc, and then subjected to the same extraction and silylation procedure as above. Analyses of the TMSi derivatives were carried out on a FID instrument using a 1.2 m X 4 mm silanized glass column packed with 3 Ofo (w/w) SE 30 on 80/100 mesh acidwashed, DMCS-treated Chromosorb W. A carrier gas (97010 N2 and 3010 H 2 ) flow rate of 50 mllmin was maintained. Injector and detector temperatures were maintained at 220°C and 240 DC, respectively. The mass spectra were determined with a LKB 9000 mass spectrometer coupled to a gas chromatograph described by BERGSTROM (1973). Helium was employed as the carrier gas at the flow rate of 25 mllmin.
Results
MS analyses showed the presence of 2,3-dibromo-4,5-dihydroxybenzyl alcohol in the marine Ascomycete and 3,5-dibromo-p-hydroxybenzyl alcohol in its culture medium together with three unidentified bromo-compounds, which were present both in the Ascomycete and its culture medium. The data for the different compounds are presented in Table 1. The mass spectra for the two bromophenols mentioned, III and V, were in entire agreement with those for authentic samples of them and for the same bromophenols isolated from red algae. Bromo-compound I contains 3 Br atoms and after silylation has a MW of 400. According to the fragmentation pattern of the mass spectra of bromo-compounds II and IV they are the same compound although with 1 and 2 Br atoms, respectively. The loss of two 0- TMSi-groups (M+-89-89) is clearly seen from their mass spectra. Their structures however remain to be elucidated. The dibromo-monohydroxy- and dibromo-dihydroxy-alcohols are known Z. PJlanzenphysiol. Ed. 82. S. 363-366. 1977.
Bromophenols in a marine Ascomycete
365
Table 1: Bromo-compounds from the marine Ascomycete and its culture filtrate. MW (Silylared)
No. of Br atoms
Ascomycete I 400 II 476 III 512 IV 552
3 1 2 2
2 or more 3 2 or more
175 0 210° 215° 220°
Culture medium I 400 V 424 II 476 IV 552
3 2 1 2
2 2 or more 2 or more
175° 203° 210° 220°
No. of TMSi groups
GLC Temp. programmed from 100 0 at 6°/min
Proposed terminal group
CH 20H
CH20H
to be esterified with sulphate at positions 1 and 4 (HODGKIN et aI., 1966; GLOMBITZA and STOFFELEN, 1972; WEINSTEIN et aI., 1975). In the present investigation no attempt has been made to establish whether the bromophenols of the marine Ascomycete occur as benzyl sulphonates. A recent investigation of the red alga Rytiphlea tinctoria (CHEVOLOT-MAGUEUR et aI., 1976) revealed lanosol as being a part of a 5,6,3',5' -tetrabromo-3,4,2',4',6-pentahydroxydiphenylmethane, which also appeared in the red pigment floridorubin as a photosynthetic pigment replacing phycobilins in this alga.
If lanosol constitutes one of the phenols of a photosynthetic pigment its appearance in a marine Ascomycete may be of interest from the point of view of the phylogeny of the Ascomycetes. Some taxonomists [d. MARTIN (1968) for references] have suggested that the Ascomycetes originate from marine red algae. Lanosol has also been identified in the two brown algae Fucus vesiculosus and Ascophyllum nodosum in very small amounts (PEDERSEN and FRIES, 1974; PEDERSEN, 1976). The possibility that marine Ascomycetes living as epi- or endophytes in these brown algae are producing these bromophenols cannot be neglected. No bromophenols have hitherto been identified in other brown or in green algae (PEDERSEN, 1976). In red algae the bromo phenols are only found in Florideae. Members of the family Rhodomelaceae are especially rich in these compounds, while they are lacking in all species belonging to the Bangioideae (PEDERSEN et aI., 1974). The reported presence of lanosol in the water of the Polysiphonia Brodiaei zone (PEDERSEN et a!., 1974), the findings of lanosol and tribromophenols in the culture filtrate of Calothrix brevissima (PEDERSEN and DASILVA, 1973) and now the four bromo-compounds in the culture filtrate of the Ascomycete indicate exudation of
z.
PJlanzenphysiol. Bd. 82. S. 363-366. 1977.
366
M. PEDERSEN and N. FRIES
these compounds surroundings.
from
the
algae
and
the
Ascomycete
into
the
immediate
Acknowledgements We thank Dr. L. FRIES for valuable advice, and Dr. A. VON HOFSTEN for having performed the electron microscopy. The investigation was supported by a grant from the Swedish Natural Science Research Council to Dr. L. FRIES.
References BERGSTROM, G.: Studies on natural odoriferus compounds. VI. Use of a pre-column tube for the quantitative isolation of natural, volatile compounds for gas chromatography / mass spectrometry. Chern. Scr. 4,135-138 (1973). CHEVOLOT-MAGUEUR, A.-M., A. CAVE, P. POTIER, J. TESTE, A. CHIARONI, and C. RICHE: Composes bromes de Rytiphlea tinctoria (Rhodophyceae). Phytochemistry (Oxf.) 15, 767-771 (1976). GLOMBITZA, K.-W., and H. STOFFELEN: 2,3-dibrom-5-hydroxybenzyl-l ',4-, disulfat (Dikaliumsalz) aus Rhodomelaceen. Plant a Med. 22, 391-396 (1972). HODGKIN, J. H., J. S. CRAIGIE, and A. G. McINNES: The occurrence of 2,3-dibromo-benzyl alcohol 4,5-disulfate, dipotassiumsalt in Polysiphonia lanosa. Can. J. Chern. 44, 74-78 (1966). KOHLMEYER, J. & E.: Is Ascophyllum lichenized? Bot. Mar. 15, 109-112 (1972). MARTIN, G. W.: The origin and status of fungi (with a note on the fossil record). In: G. C. AINSWORTH and A. S. SUSSMAN (Eds.), The fungi, Vol. III, pp. 635-648. Academic Press, New York and London, 1968. PEDERSEN, M.: Studies on marine algae with emphasis on some organic compounds produced by these algae. Acta Univ. Ups. Abstr. Upps. Diss. Sci. 355, 1-17 (1976). PEDERSEN, M., and E. J. DASILVA: Simple brominated phenols in the bluegreen alga Calothrix brevissima WEST. Planta (Berl.) 115, 83-86 (1973). PEDERSEN, M., and L. FRIES: Brominated phenols in Fucus vesiculosus. Z. Pflanzenphysiol. 74, 272-274 (1975). PEDERSEN, M., P. SAENGER, and L. FRIES: Simple brominated phenols in red algae. Phytochemistry (Oxf) 13, 2273-2279 (1974). SAENGER, P ., M. PEDERSEN, and K. S. ROWAN: Bromo-compounds of the red alga Lenormandia prolifera. Phytochemistry (Oxf) in press (1976). WEBBER, F. c.: Observations on the structure, life history and biology of Mycosphaerella ascophylli. Trans. Br. Mycol. Soc., 50, 583-601 (1967). WEINSTEIN, B., L. TRACY, R. CLAYTON, E. HARRELL, Jr., M. W. BURNS III, and J. R. WAALAND: Reexamination of the bromophenols in the red alga Rhodomela larix. Phytochemistry (Oxf) 14, 2667-2670 (1975).
MARIANNE PEDERSEN and NILS FRIES, Institute of Physiological Botany, University of Uppsala, S-751 21 Uppsala, Sweden.
Z. Pflanzenphysiol. Bd. 82. S. 363-366. 1977.