LC-SPE-NMR is a technique based on trapping the post-column analytes by solid phase extraction (SPE) and subsequent elution from the SPE cartridges using deuterated solvents [1]. It requires minimal quantities of sample and solvents, minimizes the risk of sample degradation and provides complete data sets for structure elucidation in a short time [2]. Marine microorganisms have attracted considerable attention due to their marked chemodiversity. Specimens from Asparagopsis taxiformis, a red alga collected at the rocky shore of Fortaleza beach, Ubatuba, SP, Brazil, had their surface sterilized, and were fragmented and spread on PDA plates, which led to the isolation of an endophytic fungal strain identified as Annulohypoxylon stygium (AT-03/AccessGenBank: MF062512). It was cultivated in sterilized rice medium with ultrapure water at 25ºC during 21 days. Mycelia and rice were extracted with EtOAc and the obtained crude extract, after solvent evaporation, was resuspended in EtOAc and partitioned with water. The organic phase was dried and solubilized with acetonitrile and extracted with hexane. The acetonitrile fraction (FACN) was chromatographed using a reversed phase C-18 column. Fractions FACN-F1 and FACN-F3 were analyzed by LC-SPE-NMR (C-18 column - Phenomenex Luna, 250 x 4.60 mm, 5 μm, 110Å; ACN:H2O) which led to identification of compounds 1 and 2, respectively. Compound 1 was identified as 1-phenyl-1,2-propanediol [3], a phenylalanine derivative [4], whereas 2 was identified as 8-methoxy-1-naphthol [5], which is a shunt metabolite from polyketide melanin biosynthesis. It probably results from the specific methylation of 1,8-naphthol, the polymerization precursor of melanin [6]. Analogous metabolites, such as 3,4-dihydroxy-1-tetralones and 4-hydroxy-1-tetralones, have been found in some Annulohypoxylon and Hypoxylon species, and are biosynthesized in a similar manner, by pathways branching off from the polyketide melanin biosynthesis [7]. Such features might suggest that these substances may be regarded as chemotaxonomic markers, specially because they have been identified in different species of Annulohypoxylon [6,8]. So the rapid and precise identification of these compounds in fungal extracts by LC-SPE-NMR could support the taxonomic classification, considering this genus was recently erected to accommodate species formerly included in Hypoxylon sect. Annulata J.H. Mill. emend. Y.M. Ju & J.D. Rogers [9].

References

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