New depsides from lichens: microchemical methodologies applied to the study of new natural products discovered in herbarium specimens

A high-performance liquid chromatographic method originally devised to provide a reproducible measure of retention is used to determine chemical structures of new compounds in lichens. The method relates retention to a standard homologous series so that retention indices of members of other homologous series are linear with carbon number. When two or more homologs are known, retention of new members of the same series can be accurately predicted. Combining this method with other microchemical techniques allows the determination of structures of lichen depsides in small samples of crustose species for which the total material is meager and known only from the herbarium. The conditions are sufficiently mild that chemical decompositions of labile depsides are avoided. Eight new orcinol-type p-depsides with sevenor nine-carbon oxidized sidechains on one or both rings are reported from an endemic Japanese species, Haematomma pachycarpum. The nine-carbon sidechain is the longest ever found in lichen depsides. These new compounds are higher homologs of the known lichen products confluentic, 2'-O-methylperlatolic, 4-O-methylolivetoric, and 2'-O-methylmicrophyllinic acids. In addition, an isocoumarin with a nine-carbon sidechain occurs in some specimens. Haematomma pachycarpum has the most complex secondary-product chemistry thus far reported for any lichen species. The significance of the technique used here lies in providing a way to characterize new compounds in rare or endangered species, thereby completing data sets for phylogenetic reconstructions. THE PRESENT STUDY proposes structures for eight new depsides, esters of acetyl-polymalonyl-derived phenolic acids and compounds characteristic of the lichen-forming fungi. It is based on analyses of fragments of herbarium specimens containing the new natural products. Thallus chemistry is intimately involved in the systematics of the lichen fungi (Culberson, 1969, 1970; Culberson, Culberson, and Johnson, 1977; Culberson, 1986). The microchemical methodology for the identification of several hundred known substances, perfected in a standardized method of thin-layer chromatography (TLC) (Culberson and Ammann, 1979; Culberson, Culberson, and Johnson, 1981), is now enhanced by high-performance liquid chromatography (HPLC) (Culberson, 1972; Culberson and Culberson, 1978; Strack, Feige, and Kroll, 1979; Lochmiiller et al., 1983; ' Received for publication 4 June 1986; revision accepted 18 August 1986. This work was supported in part by Grant BSR-8507848 from the National Science Foundation to Duke University. We thank J. A. Elix for samples of sphaerophorolcarboxylic acid p-methyl ether and 2'-O-methylmicrophyllinic acid, S. Huneck for a sample of glomellic acid, and H. Hertel and A. V. N. Rao for specimens. Culberson, Culberson, and Johnson, 1983; Geyer, 1985; Huovinen, Hiltunen, and von Schantz, 1985). With good techniques available for the routine identification of known compounds, we now seek ways to determine the chemical structures of new products encountered in herbarium studies. Historically, the structures of natural products of plants were established from large (commonly kilogram) quantities of material from places where the species of elaboration were abundant. The first-studied lichen products were characterized in this way as well (Zopf, 1907). Most early work focussed on major products only. Although modem chemical methodologies (nuclear magnetic resonance [NMR], mass spectroscopy [MS], and infrared red [IR] spectroscopy) require less material, the lichenologist rarely has enough to allow the necessary separations and purifications to use these techniques most effectively. Herbarium specimens, which may be as few as one for the rarest species, are the primary subject of study for the lichen systematist. But for phylogenetic reconstructions, knowledge of the chemistry of rare or endangered species and of satellite compounds is as essential as that of common species