Lichen Substances

15 Lichen Substances

CHICITA F. CULBERSON Duke

University,

Durham,

North

Carolina

27706,

USA

JOHN A. ELIX The Australian I.

II.

III.

I.

National

University,

Canberra

ACT

2601,

Australia

Introduction A. T h e f u n g a l o r i g i n o f the secondary p r o d u c t s B. M a j o r categories o f lichen p r o d u c t s Microchemical methods A. L o c a l i s a t i o n o f secondary p r o d u c t s B. Paper c h r o m a t o g r a p h y C. T h i n layer c h r o m a t o g r a p h y D. G a s c h r o m a t o g r a p h y , gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y a n d lichen mass s p e c t r o m e t r y E. H i g h performance liquid chromatography S t r u c t u r e a n d synthesis A. N e w classes o f p h e n o l i c l i c h e n c o m p o u n d s B. N o v e l substitution o f phenolic lichen c o m p o u n d s C. N e w methodology for structural elucidation D. N e w synthetic routes t o d i b e n z o f u r a n s a n d depsidones Acknowledgements References

509 511 511 512 513 514 515 516 517 520 520 523 528 529 532 532

INTRODUCTION

The secondary products of the lichen-forming fungi belong to a variety of c o m p o u n d types, some of which are not seen elsewhere in nature. These c o m p o u n d s accumulate on the outside walls of the fungal hyphae (Fig. 15.1) and are so stable in the dried lichens that even very old herbarium specimens can be used for microchemical analyses. The compounds have unquestioned systematic significance because of extensive congruences M E T H O D S I N P L A N T B I O C H E M I S T R Y Vol. 1

Copyright © 1989 Academic Press Limited

ISBN 0-12-461011-0

All rights of reproduction in any form reserved

509

510

FIG. 15.1 consocians.

C.

F.

CULBERSON

AND

J. A .

ELIX

Natural crystals of the p-depside lecanoric acid on the outside of the hyphal walls of (5000 χ ) (SEM by Dr Mason E. Hale, Jr.)

Pseudevernia

with morphology and clear ecological significance because of vital roles as antiherbivore and allelopathic agents. Some of their unique biological activities have potential medicinal value. Since the early compilation of Zopf (1907) and the later one of Asahina and Shibata (1954), numerous reviews have summarized the chemistry, occurrence and biosynthesis of lichen products (Shibata, 1963; Huneck, 1968, 1971, 1973, 1984; Culberson, 1969, 1970; Mosbach, 1969; Santesson, 1973, 1974; Culberson et al, 1977; Elix et al, 1984b). The chemistry of approximately 6000 taxa and chemotypes containing some 500 secondary products of known chemical structure have been reported in the literature. Today these numbers are rapidly increasing as analytical methods become more refined and as secondary-product chemistry continues to play a dominant role in the systematics of the lichen-forming fungi. Lichens are composite organisms consisting of a fungus and a photosynthetic partner, either a green alga or a cyanobacterium or both. The lichen's name refers to its fungal component, and approximately 18 000 species are known. T h a t the lichen fungi comprise about half of the Ascomycotina, the largest group of the Fungi, testifies to the high adaptive value of this symbiotic association with a photobiont. Typically the fungal part of the lichen consists of agglutinated hyphae of the cortex above the loosely interwoven hyphae of the medulla. Hyphae also surround the unicells

15.

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SUBSTANCES

511

of the photobiont in a layer between cortex and medulla. The photobiont provides carbohydrate and energy for the metabolic processes of itself and the fungus, while the fungus provides the physical protection of its cortex and the chemical protection of its secondary products. A.

The Fungal Origin of t h e Secondary Products

All of the secondary substances for which lichens are famous are of fungal origin. Because cultures of lichen fungi grow very slowly and may fail to show the products characteristic of mature thalli in nature, nearly all chemical data come from studies of natural lichens. These natural materials, which may have accumulated the compounds over decades, need only be extracted with a suitable organic solvent to yield a b u n d a n t residues for analysis. Hypothesised biosynthetic pathways to lichen products come from : (1) analogies to pathways in non-lichen fungi (Turner, 1971; Turner and Aldridge, 1983); (2) a few experimental studies on lichens themselves (e.g., Mosbach, 1964; Yamazaki et al., 1965; Yamazaki and Shibata, 1966; Mosbach and Schultz, 1971); (3) observed joint occur­ rences of compounds; and (4) laboratory interconversions and biomimetic syntheses (Elix and Gaul, 1986; Elix et al. 1987a). Most of these secondary products are clearly acetyl-polymalonyl-derived, but a few come from the shikimic and mevalonic acid pathways. B.

M a j o r Categories of Lichen Products

The principal classes of lichen products are given in Table 15.1 below by biogenetic similarity with the numbers of compounds of known structure indicated (in paren­ theses). Of the a c e t y l - p o l y m a l o n y l - d e r i v e d c o m p o u n d s , aromatic products of polyketide T A B L E 15.1.

M a j o r classes o f secondary p r o d u c t s i n lichens.

I. A c e t y l - p o l y m a l o n y l p a t h w a y A . Secondary a l i p h a t i c acids, esters a n d related c o m p o u n d s (42) B. P o l y k e t i d e - d e r i v e d a r o m a t i c c o m p o u n d s 1. M o n o n u c l e a r p h e n o l i c c o m p o u n d s (17) 2. D i - a n d t r i - a r y l derivatives o f simple p h e n o l i c u n i t s a. p a r a - D e p s i d e s , tridepsides, b e n z y l esters a n d weta-depsides (133) b. D e p s i d o n e s , depsone a n d related d i p h e n y l ethers (88) c. D i b e n z o f u r a n s , usnic acids a n d related c o m p o u n d s (17) 3. C h r o m o n e s (13) 4. N a p h t h o q u i n o n e s (4) 5. X a n t h o n e s (26) 6. A n t h r a q u i n o n e s a n d biogenetically related x a n t h o n e s (54) I I . Mevalonic acid pathway A . D i - , sester- a n d t r i t e r p e n o i d s (62) B. Steroids (20) I I I . S h i k i m i c acid p a t h w a y A . T e r p h e n y l q u i n o n e s (2) B. P u l v i n i c a c i d derivatives (12)

512

C.

F. C U L B E R S O N

A N D J. A .

ACETYL-POLYMALONYL

DERIVED /S-ORCINOL TYPE

ORCINOL TYPE C 5H1 1

ELIX

CH3

OH

H 3C

OH

HO ^ > - C O O ^ > - C O O C H 3 OH

1.

2.

ALIPHATIC ACID BOURQEANIC ACIO

HOOC / \

Η (CH )

3

2 12

4.

Ο

rw_

nu

5.

ATRANORIN

OH

>w

OHC

ALECTORIAUC ACIO

3 ° ^ ( Q > - C 0^ 0^ C5 H1 1

C 3H ° ^ ^ > - C O O ^ ^ O H

CH3

HOOC

Y ^ O ^ C H , Cl 7. C H R O M O N E

8.

0.

META-DEPSIDE

OH

>"'3

/

° C° " v O / "

Η 30 / θ /

C OH O

COOH

HO

CHO

META-DEP8I0E

pOCCH-CHCOOH CHgOH

CH3

OH

H 3C

THAMNOLIC ACIO

MEROCHLOROPHAEIC ACIO

SORDIOONE

C H 3 0 A ^ sV0 ' ^ - ^ O C Η

H

QYROPHORIC ACIO

HO

Ο

r

β. BENZYL ESTER

TRIDEPSIDE

C H

P i

OH

H O - ^ C O O C H 2 ^ C H 3

Ο

CH.

1 0.

PARA-0EP8I0E

HOOC

(+)-PROTOLICHESTERINIC ACIO

H,C

OH

3.

H O ^ > C O O ^ > C O O ^ O > C O O H

FATTY ACIO

HO

OHC

PERLATOLIC ACIO

CH, \ °

/

CgH^

PARA-0EP8IDE

c o o -

HO-(Qy

(Oy

C OH O

3

1 1.

XANTHONE

1 2.

DEP8IDONE

QRAYANIC ACIO

LIC HE XANTHONE

OEPSIOONE

FUMARPROTOCETRARIC ACIO CHXO

Η r ° v ^ V ^ X - r) ' C

O3

C

H O ^ ^ o ^ ^ O H 1 3.

1 4.

NAPHTHOQUINONE

DIBENZOFURAN

Ο

OH

CH

•P COCH,

'

1 5.

USNIC

ACID

STREP3ILIN

USNIC ACIO

SHIKIMIC ACID DERIVED

MEVALONIC ACID DERIVED

RHOOOCLADONIC ACID

HO

lo

C H 20 £ H2 '

CH H3 5 3

H

.—

v

COOH ΟΗ

ο 1 6.

ANTHRAQUINONE PARIETIN

ο 1 7.

PULVINIC ACID DERIVATIVE PULVINIC ACIO

OH

1 8.

TRITERPENE LEUCOTYLIN

FIG. 15.2. Examples of the major categories of secondary lichen products. (From Culberson, 1986.)

origin are especially well represented, the most characteristic being formed by the union of two or three simple orcinol or β-orcinol-type phenolic units through ester, ether and carbon-carbon linkages (Fig. 15.2). In the biogenetic outline above, these most characteristic products are di- and tri-aryl derivatives of simple phenolic units (category LB.2). Specific compounds in other categories may be unique to lichens but are often very closely related to products in non-lichen fungi. In addition to the compounds of known chemical structure, many of unknown structure have been given common names and assigned to compound class, because they are frequently encoun­ tered and easily recognised by microchemical methods.

II.

MICROCHEMICAL

METHODS

Although most of our knowledge of the structures of lichen products comes from study

15. LICHEN SUBSTANCES

513

by both traditional and modern chemical methods, the extensive data on the natural occurrence of these compounds are based almost entirely upon microchemical analyses. The earlier microchemical methodologies, briefly summarised here, are reviewed by Santesson (1973) and by White and James (1985). Broad surveys using extracts from fragments of herbarium specimens began after the 1930s when Yasuhiko Asahina invented simple microcrystal tests to identify specific compounds, most of them of known chemical structure and localised in specific histological regions of the thallus. A.

Localisation of Secondary Products

Pigmentation patterns and thallus spot tests gave the first evidence that lichen products were not uniformly distributed throughout the thallus. The yellow pigment usnic acid and many brilliantly coloured anthraquinone derivatives were obviously restricted to the upper cortex in many species. M a n y of the depsides and depsidones, which are colourless, were early demonstrated to be localised in the medulla by thallus colourtest reagents. /.

Microchemical

tests applied to sectioned

thalli

The c o m m o n colour-test reagents (listed in Table 15.2 below) applied to the cortex and medulla for routine identifications of lichen specimens, can also locate compounds in sectioned thalli. These tests can give clues to the chemical nature of the secondary products as well. TABLE 15.2.

Reagents for thallus spot tests.

C = Either saturated aqueous Ca(OCl) 2 or common household bleach (NaOCl) turns (1) red with m-dihydroxy phenols, except those substituted between the hydroxyl groups with either —CHO or —COOH and (2) green with the dihydroxy dibenzofuran strepsilin. Κ = 10% KOH turns (1) yellow to red with most o-hydroxy aromatic aldehydes, and (2) bright red to deep purple with anthraquinone pigments. KC = The Κ reagent followed by the C reagent turns (1) red with C-negative depsides and depsidones that hydrolyse rapidly (often due to participation by a keto group at the a- or β-carbon of a side chain ortho to the ester linkage) to yield a w-dihydroxy B-ring unit, (2) blue with strepsilin, and (3) yellow with usnic acid. PD = Alcoholic p-phenylenediamine turns yellow, orange or red with all aromatic aldehydes.

Microcrystal tests that depend upon rapid precipitation of a slightly soluble deriva­ tive can also locate specific compounds in sections. For example, an early study by Asahina (1936) showed the distributions of (1) evernic acid in the medulla of Evernia mesomorpha Nyl. by precipitation of its barium salt and (2) usnic acid in the cortex and diffractaic acid in the medulla of Alectoria ochroleuca (Hoffm.) Mass. by precipitation of their sodium salts. A solution of K O H and K 2 C 0 3 was used to locate norstictic acid in the hymenium of Letharia californica (Lev.) H u e (W. L. Culberson, 1969a).

514 2.

C.

F.

CULBERSON

Scanning electron microscopy

AND

J. A .

ELIX

and laser microprobe mass

spectrometry

Early scanning electron microscopy (SEM) studies showed conspicuous crystals on the cortex and in the medulla of several species known from microchemical tests to contain high concentrations of lichen products in these tissues (Fahselt et al, 1973; Peveling, 1973; Hale, 1973). But some crystals seen by SEM were one of several crystal forms of calcium oxalate rather than phenolic products. Mathey and Hoder (1978) used energydispersive X-ray spectrometry (EDX) and cathodoluminescence (CL) to identify crys­ tals in 40 pm thick sections of four lichens known to contain UV-fluorescent chlorinated xanthones. Lecanora cerebellina Poelt, which contains the chlorinated xanthone vinetorin, showed two morphologies of crystals in the cortex. Luminescent needles showing a signal for chlorine by E D X were identified as vinetorin, and non-luminescent crystals of a different morphology giving a strong signal for calcium and little chlorine were tentatively identified as calcium oxalate. Later Jackson (1981) demonstrated that pruina on thallus surfaces of several species was due to crystals of calcium oxalate. X-Ray diffraction analysis of a crystal picked from the surface of Pyxine caesiopruinosa (Nyl.) Imsh. confirmed that the very abundant eight-sided bipyramidal crystals were calcium oxalate dihydrate and not the secondary product (lichexanthone) known in this species. Confirmations of crystal identifications by mass spectrometry have been made with an instrument that combines a light microscope and a microprobe mass spectrometer with a lateral resolution of about 1 μηι. Ionisation is laser induced, making it potentially applicable even to thermally labile compounds. Laser microprobe mass spectrometry (LMMS) of solid inclusions in a thallus cross-section of Laurera benguelensis (Mull. Arg.) Zahlbr. confirmed lichexanthone by the major peak at mjz 287 (M + H + ) in the positive-ion spectrum (Mathey, 1981). More recently, L M M S has been coupled with fluorescence microscopy and transmission electron microscopy to locate compounds in semithin sections of several species (Mathey et al., 1987). The point analysed on a thallus section by the laser microprobe was examined in detail by transmission electron microscopy. For example, base peaks at mjz 271, 297 and 313 came from orange extrusions from the hyphae of Phaeographina chrysocarpa (Raddi) Redinger. Lichexan­ thone and russulone, a new tetracyclic anthraquinone, were located in different zones of the fruiting body of Lecidea russula Ach. B.

Paper C h r o m a t o g r a p h y

Using Asahina's microcrystal methods, herbarium botanists discovered extensive corre­ lations between the chemistry, morphology and geography of lichens. This simple technique required no special equipment, and experience yielded generally accurate analyses of major products. Nevertheless, it soon became clear that the microcrystal tests could detect only a small subset of the lichen products and were inadequate for the study of mixtures. The limitations of the microcrystal tests, however, became irrelevant with the advent and refinement of the methods of chromatography. In 1952 Wachmeister introduced paper chromatography to identify 10 of the 25 thenknown phenolic acid units formed by hydrolysis of lichen depsides. Later, he extended the method to naturally occurring depsides, depsidones, pulvinic acid derivatives and usnic acid (Wachtmeister, 1955, 1956). Paper chromatography proved that the chemistry of many species was more complex than had been suspected from either

15.

LICHEN

SUBSTANCES

515

macroextractions or microcrystal tests. The long analysis times, poor spot resolutions and low sensitivities were overcome by the development of thin layer chromatography. C.

Thin Layer C h r o m a t o g r a p h y

Thin layer chromatography (TLC) is now the most widely used microchemical method for identifying lichen products. Early studies sought useful solvents and visualising agents and demonstrated the technique for a wide variety of c o m p o u n d s (Santesson, 1965, 1967; Bendz et al., 1965, 1966), allowing a rapid expansion of the chemical database for the lichens. But different workers rarely used the same solvent systems and chromatographic conditions, and their results were not comparable. This problem was solved when pre-spread T L C plates became commercially available, making a standardised method possible. 1.

A standardised

method

A standardised method, which with only slight modifications is still in general use, employs three solvent systems (designated A, Β and C) and two internal controls (atranorin and norstictic acid) to which all R{ data are compared (Culberson and Kristinsson, 1970; Culberson, 1972b, 1974; Culberson and A m m a n n , 1979; Culberson et al., 1981; Culberson and Johnson, 1982; White and James, 1985). For each solvent system, a spot is assigned to an R{ class determined by its position relative to the internal controls. D a t a on punched cards or the computer are then sorted to find all compounds with the same R{ classes. Of these possibilities, those with similar spot characteristics (colour, fluorescence, etc.) are compared chromatographically to the unknown. Additional solvent systems and visualising agents are available for com­ pounds that d o not separate well in the initial analysis, and two-dimensional T L C is used for complex mixtures (Maass, 1975; Culberson and Johnson, 1976). 2.

Solvent systems for separations on silica gel plates

Solvent A = Toluene-dioxane-acetic acid (180:45:5) owes its distinctive characteristics to the ability of dioxane to associate with phenolic hydroxyls. Solvent Β = Hexane-methyl tert.-butyl ether-formic acid (140:72:18) is a more stable, less volatile and safer version of the original solvent Β ( = hexane-diethyl ether-formic acid; 130:80:20), and gives good separation of c o m p o u n d s that differ only slightly due the length of side chains or the number of C-methyl substituents. Solvent C = Toluene-acetic acid (170:30) is an excellent general solvent for a wide variety of compound types. Solvent Ε = Cyclohexane-ethyl acetate (75:25) is a new solvent used in the revised method of Elix et al. (1988) described below. It is recommended for less acidic compounds that give high R{ values in solvents A, Β and C (e.g., methyl esters such as atranorin and chloroatranorin, decarboxylated depsidones such as pannarin, the terpenoids and many pigments including the usnic acids and xanthones).

516

C.

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CULBERSON

AND

J. A .

ELIX

Solvent G = Toluene-ethyl acetate-formic acid (139:83:8) is useful for β-orcinol depsidones and other compounds with low R{ values in solvents A, Β and C (Culberson et al, 1981). 3.

Visualisation and quantification

of spots

A significant feature of T L C is the broad range of spot characteristics that can be used in addition to R{ data. Before the T L C plate is sprayed, each spot is examined for colour and for fluorescence or quenching under short- and long-wave UV. F o r general screening, spots are then visualised with 10% H 2 S 0 4 and heat (110°C for 15-30 min). Although H 2 S 0 4 charring is not so sensitive for some classes of compounds, it does detect the broadest range of compound types including many aliphatic substances in addition to the common phenolic ones. When the d a m p chromatogram is allowed to partially dry at room temperature before being heated, even fatty acids, often not detectable with UV or charring, appear as opaque spots against the translucent background. After the plates have been charred, the compounds give a range of visible colours and some even have a characteristic fluorescence shortly after being removed from the oven (White and James, 1985). Special reagents are available for compounds difficult to detect with general acid charring. Examples of alternative reagents with greater selectivity or sensitivities for certain phenolic lichen products are: (1) anisaldehyde/sulphuric acid and heat (110°C) (Leuckert, et al, 1979); (2) 3-methyl-2-benzothiazolone hydrazone hydrochloride (MBTH) (Archer, 1978); (3) fast Bordeaux BD salt followed by sodium carbonate (FB) (Maass, 1975); and (4) a stabilized P D reagent (Steiner, 1955). Valuable spectral data can be obtained from T L C spots (Leuckert et al, 1979, 1981; Leuckert and Meinel, 1981; Leuckert, 1984; Leuckert and Mayrhofer, 1985). Absorp­ tion maxima are measured with a chromatogram spectrophotometer and comparisons made to the spectra of controls chromatographed on the same plate. C h r o m a t o g r a m scanners have been used to quantify the secondary products in individuals from different parts of a species' geographic range (W. L. Culberson, 1969b), in lichens maintained under artificial conditions (Fahselt, 1981a) and in experimental transplants (Fahselt, 1981b). 4.

Computer-assisted

identifications

Recently, a standardised T L C method has been designed to take advantage of computer technology (Elix et al, 1988). It uses six solvent systems and eight control compounds. Relative R{ values are used to sort a computerised database. The program can also list biosynthetically related compounds as an aid to the identification of satellite substances. D.

Gas C h r o m a t o g r a p h y , Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y and Lichen Mass S p e c t r o m e t r y

The typical lichen depsides and depsidones have thermally labile ester linkages, and techniques that require volatilisation give decomposition products that can confuse the analysis. G a s chromatography-mass spectrometry (GC-MS) is widely used in the perfume industry for the constituents of odoriferous fractions of extracts of Oakmoss, the lichen Evernia prunastri (L.) Ach. Such fractions are already highly degraded,

15.

L I C H E N SUBSTANCES

517

having been isolated by steam distillation and alcohol extraction. The degradation products of depsides, especially those formed by alcoholysis, are readily identified by this method (ter Heide et al, 1975). Xanthones (Santesson, 1969), anthraquinones (Santesson, 1970) and usnic acid lack the labile ester linkage and have been successfully studied by G C or lichen mass spectrometry (LMS). Fahselt (1975) analysed the cortical pigment usnic acid in Xanthoparmelia cumberlandia (Gyeln.) Hale using an OV-17-coated C h r o m o s o r b W (80/100 mesh) column with a gradient from 160 to 260°C and a flame ionisation detector. Santesson (1967) studied xanthone pigments and obtained mass spectra by introducing small lichen samples (some < 5 0 n g ) into the direct inlet system. The xanthones sublime as the temperature is raised (100-150°C) under very low pressure. Although low-mass decomposition products were evident, the xanthones generally gave prominent molecular ions, and the spectra of mixtures could often be seen as additive of the individual components. The spectra of some other compounds—such as usnic acid, pulvinic acid derivatives and dibenzofurans—were more complex but still sufficiently distinctive to allow identifications in some lichens. Even though some chemical structures of xanthone pigments proposed then have since been corrected, these early studies clearly demonstrated both the potential power and the limitations of L M S . E.

High P e r f o r m a n c e Liquid C h r o m a t o g r a p h y

All of the phenolic lichen products, including those that are nonvolatile or too thermally labile to be analysed by G C , are ideally suited for study by high performance liquid chromatography (HPLC). Early attempts, using normal-phase silica columns with mobile phases of hexane-isopropyl alcohol-acetic acid (Culberson, 1972a) or 2,2,4trimethylpentane-chloroform-acetic acid (Nourish and Oliver, 1976), showed the poten­ tial for detecting and quantifying lichen products. In general, however, the separations were not significantly better than those obtained by T L C . In the late 1970s bonded reversed phase columns were developed, providing a powerful H P L C method that also better complements the normal phase T L C techniques. 1.

Isocratic

elution

The first applications of reversed phase conditions separated orcinol and β-orcinol depsides on C18 columns with methanol-water-acetic acid solvent systems (Culberson and Culberson, 1978; Culberson and Hertel, 1979; Culberson et al, 1979; W. L. Culberson and Culberson, 1978, 1981). The same solvent systems are useful for orcinoltype depsidones and dibenzofurans related to didymic acid. Other studies substituted 0r//z0-pfiosphoric acid for acetic acid in the mobile phase to analyse diploschistesic a c i d lecanoric acid mixtures (Lumbsch and Elix, 1985) and depsidones related to pannarin (Elix et al, 1986). Polar dibenzofurans and many β-orcinol depsidones give better results by gradient elution. 2.

Gradient

elution

Gradient methods are excellent for H P L C analyses of crude lichen extracts, which often contain compounds of wide-ranging hydrophobicities. Strack et al (1979) separated 13 selected lichen products on a LiChrosorb RP-8 column with a 70 min linear gradient

518

C F . CULBERSON AND J. A. ELIX

Column Pocking :

LIChrosorb RP-8(5um) Column Dimension : 4x250 mm Solvent A: 2•/• Acetic Acid Methanol Solvent B : Gradient Profile : Linear in 70 min from A to B Flow Rate : 1 ml min 1 Sample Size : 10 u.1

FIG. 15.3. Gradient HPLC of a representative mixture of phenolic lichen products including examples of depsides, depsidones, dibenzofurans and a pulvinic acid derivative. (From Strack et al, 1979).

from water containing 2% acetic acid to 100% methanol and UV detection at 254 nm (Fig. 15.3). Although the depsidone physodic acid did not separate from the yellow pulvinic acid derivative leprapinic acid, the latter was detected without interference by monitoring the absorbance at 365 nm. The long analysis time could be shortened for specific applications. A 30 min linear gradient from 0.5% acetic acid in water to 100% methanol was used to detect six known (constictic, stictic, norstictic, psoromic, gyrophoric and rhizocarpic acids) and seven unidentified components in the Rhizocarpon superficielle group (Geyer et al, 1984). Similarly, a 20 min linear gradient from 30% methanol/1% or^o-phosphoric acid to 100% methanol resolved two depsides (lecanoric acid and erythrin), a dibenzofuran (schizopeltic acid) and traces of several satellite compounds (Follman and Geyer, 1986). Feige et al. (1986) used the same solvents with a 20-min gradient from 0% to 100% methanol (0.7 ml min" 1 ) and a Nucleosil-5 C 8 (25 cm x 4 mm) column to study similar major products in Roccella hypomecha (Ach.) Bory. Under these conditions, Iecanoric and schizopeltic acids were well resolved, allowing the detection of an unidentified compound of intermediate mobility. Huovinen et al (1985) developed a gradient method to detect and quantify 41 secondary products of Cladonia and Cladina. The methanol-dilute orf/zö-phosphoric acid (0.09 g of 85% phosphoric acid diluted to 100 ml H 2 0 , pH 2.0) solvents were varied along an 85 min linear gradient from 20% to 99% methanol at 1.0 ml min" 1 on a 5 jam LiChrosorb RP-8 column (25 cm x 4 mm). For an analysis of minute single-spore

15. LICHEN SUBSTANCES

519

cultures, Culberson et al. (1988) used an Ultrasphere C 1 8- c o l u m n (25 cm χ 4.5 mm) and a non-linear gradient of methanol-water-acetic acid specifically optimised to separate all the major depsides and depsidones of the chemotypes of Cladonia chlorophaea (Florke ex Somm.) Spreng. Most workers using H P L C for lichen compounds have combined the technique with T L C and/or M S to verify identifications of the peaks. This is fortunate because the unique chemistry of reversed phase separations and the high sensitivity of U V detectors have revealed many compounds new to lichens. Nevertheless, verification of the identity and purity of peaks remains a major problem for screening large numbers of specimens. In an effort to address this problem, Huovinen et al. (1985) used a retention index (see Section ILE.4. below) and dual detection at 254 and 270 nm. F r o m the absorbance data for each compound of interest and for a standard concentration of benzoic acid in the same analysis, an absorbance ratio (A254/A270 was calculated by dividing the ratio of peak heights (in mm) at 254 nm by the ratio of peak areas at 270 nm. This absorbance constant is a characteristic of the compound that varies if impurities with different absorbance characteristics co-chromatograph. 3.

Quantitative

analysis

H P L C has been used to measure either absolute or relative concentrations of lichen compounds: (1) in individuals of a species from different parts of its geographic range (Huovinen, 1985); (2) within and between single thalli or thallus clumps (Stephenson and Rundel, 1979; Archer, 1981; Fahselt, 1984; H a m a d a , 1984; Huovinen, 1985; Huovinen and Ahti, 1986); and (3) in cloned cultures as a function of age, temperature and light (Culberson et al., 1983). Two studies systematically examined sources of error, including those introduced during the preparation and extraction of lichen samples. Huovinen et al. (1985) recommended that specimens be dried at room temperature for several weeks, uniformly pulverised, stored over silica gel for at least two days and extracted with a mixed solvent of acetone-ethyl acetate-dimethylformamide (40:40:20; v/v/v). F o u r 3 min extractions at 50°C were sufficient to remove even very slightly soluble products. An aliquot of the centrifuged and filtered extract could be chromato­ graphed directly without concentration. A similar study by Geyer (1985) found that of six different extraction solvents tested on lichens containing a variety of compound types, acetone (1 ml per 10 mg of dry lichen) for one hour at room temperature provided nearly complete extraction. 4.

Retention

indices and correlations with chemical

structure

Retention times (/ R) vary somewhat with even slight changes in solvent composition and appreciably as columns age. Methods that convert / R or capacity factors (kf) to retention indices (R.I.) can give values that are quite constant over the lifetime of a column and for modest changes in solvent composition. Two retention index methods have been used for lichen products: the first calculates a value (R.I.) relative to two internal controls and the second calculates a value (/) relative to a standard homologous series. The method described by Huovinen et al. (1985) for a gradient analysis of secondary products of Cladonia and Cladina used internal standards of low (benzoic acid) and high

520

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CULBERSON

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J. A .

ELIX

(bis-(2-ethylhexyl) phthalate) retention. Retention index values (R.I.) were defined as: R.I. =

/ R of the compound — tR benzoic acid / R bis-(2-ethylhexyl) phthalate — tR benzoic acid

These values were more stable than retention times and were used to form a database. The second method, which is better adapted to isocratic H P L C , uses the logarithmic increase in k' (capacity factor) with increasing carbon numbers in hydrophobic series. The retention index (I) based upon this relationship was developed by Baker and M a (1979) and demonstrated for urushiol homologues in species of Rhus (Ma et al, 1980). To standardise H P L C data, / values of compounds are calculated relative to a control series of 2-ketoalkanes for which the values are defined as equal to the carbon number χ 100. Because the calculation uses log k' rather than retention times, / also has a linear relationship to carbon number for any new series of homologous compounds encoun­ tered. For the study of orcinol-type depsides, depsidones and dibenzofurans, / values are conveniently plotted against the number of side chain carbons (Fig. 15.4). Studies have shown that / does not vary appreciably with small changes in chromato­ graphic conditions or column age, making it ideal for databases and comparisons from different laboratories (Smith, 1984; Smith et al, 1986). Additionally, as values for known compounds accumulate, calculated values for new but chemically related ones can be included. This method has provided an instant first clue to the identity of new natural products in lichens (Culberson et al, 1984, 1985, 1987). Because of the expense and technical complexity of H P L C and especially of gradient elution methods, T L C will probably continue to be more widely used for routine identifications. Nevertheless, H P L C is the method of choice for detecting trace satellite compounds, analysing very small samples, quantifying lichen products, and providing structural information from retention characteristics.

III.

STRUCTURE AND

SYNTHESIS

Since the last major reviews of the chemistry of lichen substances by Huneck (1984) and Elix et al. (1984b) this field has continued to develop apace. Several new classes of phenolic compounds have been discovered in lichens during the intervening period and these are discussed below. In addition, improved methods for the purification and the structural elucidation of lichen phenolics have been reported. In particular the develop­ ment of sophisticated techniques in carbon-13 and proton N M R spectroscopy and mass spectrometry have greatly aided these structural studies. Biomimetic-type syntheses have been developed for a number of lichen compounds, providing efficient routes to these natural products as well as new insights to the biosynthesis of these compounds. A. /.

N e w Classes of Phenolic Lichen Compounds A lichen

biphenyl

The major component of the lichen Psoroma contortum Nyl. has been identified as the unique biphenyl, contortin (1). The structure of this c o m p o u n d followed from

15.

LICHEN

521

SUBSTANCES

2000 Γ

2

4

6 NUMBER

8 OF

10 SIDECHAIN

12

14

16

18

CARBONS

FIG. 15.4. The linear relationship of retention index and number of sidechain carbons in homologous series of naturally occurring orcinol depsides and their hydrolysis products. (From Culberson et al, 1987.)

the spectroscopic properties and was confirmed by total synthesis (Elix et al., 1984a) as outlined in Fig. 15.5. Thus Ullmann condensation of 4-benzyloxy-3-iodo-2,6-dimethoxytoluene (2) gave the biphenyl (3). Subsequent bromination of (3) with Nbromosuccinimide afforded the d i b r o m o derivative (4), which on reaction with cuprous cyanide yielded the dinitrile (5). Treatment of (5) with methyllithium and subsequent hydrolysis led to the diketone (6) and ultimate deprotection by hydrogenolysis over palladised carbon gave contortin (1). Contortin (1) appears to be derived biosynthetically by phenolic coupling of two methylphloroacetophenone moieties, and hence would appear to be related to both usnic acid (7) and its derivatives as well as the diphenyl ether, leprolomin (8) (Elix et al., 1978).

522

C. F. CULBERSON A N D J. A.

ELIX

OCHoPh Copper

H 3C O " Y ^ O C H

OCH3

*

3

J

180°

CH 3

H 3C

2 P h C H 20

4 5

6

S

3

O C H 2P h

ΗΧΟ-^ί—^~OCH H3CH3CO

H 3CO O C H 3 C H 3

^

MeOC H2/Pd-C 3

*

OH HO

MeO

O C H 3C H 3

COMe OMe

Me

R = Br R = CN R = COCH3

OMe OMe Me 1

,COCH, H,C COCH 3

FIG. 15.5.

2.

MeO

Total synthesis of contortin (1).

Metal-ligand

>

(n) CuCn (iii) MeLi

complexes

Phenolic lichen acids have long been implicated as assisting in the chemical weathering of rocks. These lichen compounds are to some extent soluble in water, due to the presence of multiple polar functionalities (e.g. O H , C 0 2 H groups). Moreover, soluble metal complexes may be formed when such lichen compounds react with suspensions of minerals and rocks (Huneck, 1966; Iskander and Sayers, 1971, 1972; Ascaso and Galvan, 1976; Ascaso et al, 1976). Although lichens often contain quite high quantities of metals (for instance lichens growing on iron-rich rocks have been reported (Lange and Ziegler, 1963) to contain from 6000 to 16 0 0 0 p g g _ 1) whether they were simply accumulations of inorganic minerals or metal-ligand complexes has not been clarified until recently. Certainly the former was known to be true—for instance ferric oxide was identified in the ochraceous crust of Acarospora sinopica (Wahlenb.) K o r b . (Weber, 1962) and aluminium-containing goethite in the ferrugineous crust of Tremolecia atrata (Ach.) Hertel (Jones, et al, 1981). Recently the localisation of norstictic acid and copper in green, copper-rich specimens of Acarospora smaragdula (Wahlenb.) Massal. and Lecidea lac tea Florke ex Schaer. from cupriferous substrata was studied by optical and scanning electron microscopy and by electron probe microanalysis (Purvis et al, 1987). Infrared spectroscopy of the lichen material and a synthetic copper-norstictic acid complex provided evidence that such complexing occurred within the cortex of these lichens and led to their unusual surface coloration. The synthetic copper-norstictic acid-dmf complex (9) was prepared by reaction of copper (II) acetate monohydrate with two moles of norstictic acid in

15.

L I C H E N SUBSTANCES

523

OH HO

Ο

CH3

9

FIG. 15.6. Structure of copper-norstictic acid-
warm dimethylformamide and was formulated as illustrated in Fig. 15.6. Preliminary observations have also indicated that copper may form a complex with psoromic acid and colour the lichen thalli green (Purvis et al, 1987). B.

Novel S u b s t i t u t i o n of Phenolic Lichen C o m p o u n d s

1.

Depsides

Given the large number of lichen depsides known (Huneck, 1984; Elix et al, 1984b) it seems surprising that new compounds with novel structural features continue to be isolated. M o r e recently these have concerned the length of the alkyl side chains, with compounds such as superlatolic acid (10), isolated from Micarea prasina Fr. (Elix et al., 1987g) and Dimelaena oreina (Ach.) N o r m . (Culberson et al., 1984) and comprising two nuclei with saturated C 7 side chains, and more recently to compounds such as 2-0methylsuperphyllinic acid (11) from Haematomma pachycarpum (Mull. Arg.) Zahlbr. and glaucophaeic acid (12) from Porpidia glaucophaea (Korb.) Hertel and K n o p h , where both of the component nuclei have oxidised C 9 side chains (Culberson et al., 1987) (Fig. 15.7). Another recent discovery was that in solution, ra-scrobiculin (13) was in dynamic equilibrium with the corresponding / w # - i s o m e r , /7-scrobiculin (14) (Elix and Gaul, 1986). This equilibration provides compelling evidence that meta-depsides arise biosynthetically by C-hydroxylation of the B-ring of a /?#ra-depside followed by acyl migration between the ortho-oxygen functionalities to give the thermodynamically more stable meta-isomer. The co-occurrence of closely related para- and weta-depsides such as divaricatic acid (15) and sekikaic acid (16) has been observed in a number of Ramalina spp., providing further circumstantial evidence for this proposal (Elix and Gaul, 1986). However, confirmation of this proposal must await feeding experiments with suitably labelled precursors. 2.

Diphenyl

ethers

The majority of lichen diphenyl ethers are biosynthetic precursors for, or catabolites of> similarly substituted depsidones (Huneck, 1984; Elix et al, 1984b). Two such diphenyl

524

C.

F.

CULBERSON

AND

J.

A.

ELIX

R

10 11 12

R = C 7H 1 5, RT = H, R 2 = Me, R 3 = Η R = CH 2COC 7H 1 5, RT = H, R 2 = Me, R 3 = Me R = CH 2COC 7H 1 5, RT = Me, R 2 = H, R 3 = Me

FIG. 15.7.

ethers, epiphorellic acid 1 (17) and epiphorellic acid 2 (18), have recently been isolated from Cornicularia epiphorella (Nyl.) D u Rietz by Garbarino and colleagues (Fiedler et al, 1986). The latter compound (18) is the first diphenyl ether known to contain a γketoalkyl side chain where the ketone group is not implicit in the acetate-polymalonate pathway to this compound, although two depsides with such side chains are known (e.g. miriquidic acid and normiriquidic acid) (Fig. 15.8).

18

R = CH 2CH 2COC 2H 5

2 0 R = OMe

FIG. 15.8.

Two uniquely substituted diphenyl ethers, micareic acid (19) and methoxymicareic acid (20), were isolated from chemical races of the lichen Micarea prasina Fr. (Elix et al, 1987g). The structure of these compounds was confirmed by total synthesis as outlined in Fig. 15.9. The key steps involved Ullmann-like condensation between the 3-chloro-2enone (21) and the phenols (22) and (23), and subsequent aromatisation of the enol ethers (24) and (25) (Elix et al, 1987g). The ethers (19) and (20) have a distinctly different substitution pattern, and presum-

15.

٩

A 7 - C 7H 15

A 7 - C 7H 15

0Ο 2Μβ

R v

K 2C 0 3

H O ^ ^ 22 R = Η 23 R = OMe

n-C 7H 15

A ? - C 7 H 15

^ X . C 0 2M e

Κ γ Α ^ Χ 0 2Μ β OMe

CI

21

525

LICHEN SUBSTANCES

/?-C 7H 15

X . C 0 2M e

C7H15

X 0 2M e

(i) B r 2, A c 20 (ii) A c O H , H 20

OMe 24 25

y A ^ C 0 2M e OMe

HO

R=Η R = OMe

Mel K 2C 0 3

A 7 - C 7H 15 19

20

(')

KOH

Vo H

+

/?-C 7H 15

BCI3

<

MeO

X)Me

FIG. 15.9. Synthesis of micareic and methoxymicareic acids.

ably biosynthetic origin, to other known diphenyl ethers. As the structurally related depside superlatolic acid (10) was also found to occur in M. prasina, it was suggested that (19) may arise by an enzymatically induced Smiles rearrangement of the depside (10) (Elix et al, 1987g). Indeed, when methyl superlatolate (26) was treated with sodium hydride in dimethylformamide, methyl micareate (27) was formed (Fig. 15.10). This synthesis gives credence to the proposal that a Smiles rearrangement of the appropri­ ately substituted depside provides a viable synthetic pathway to these diphenyl ethers.

FIG. 15.10.

Biomimetic-type synthesis of methyl micareate.

526

C.

F.

CULBERSON

AND

J. A.

ELIX

FIG. 15.11.

3.

Lichen

xanthones

Lichens are known to produce many xanthones (Elix et al., 1984b; Culberson, 1969, 1970; Culberson et al., 1977), all of which are derivatives of norlichexanthone (28) (Fig. 15.11). It has been suggested that the biosynthesis of these metabolites involves the cyclisation of a single, linear polyketide chain (Culberson, 1969; Hill et al., 1982) which undergoes ring closure to give directly (possibly via the intermediacy of a benzophenone which subsequently cyclises) the typical norlichexanthone substitution pattern. By contrast, many xanthones isolated from higher fungi exhibit a substitution pattern like that of ravenelin (29) or its derivatives (Hill et al., 1982). In this case the cyclisation of the linear polyketide chain involves the intermediacy of an anthrone or anthraquinone, with subsequent oxidative cleavage giving the intermediate benzophenone which cyclises to form a xanthone (Fig. 15.12). Thiomelin (30) and a number of its congenors (32-35) isolated from the lichens Rinodina thiomela (Nyl.) Mull. Arg. and Rinodina lepida (Nyl). Mull. Arg. have now been shown to have the ravenelin substitution pattern (Elix et al, 1987d). The crystal structure of thiomelin diacetate (31) was determined by X-ray diffraction, while that of the congenors, 8-O-methylthiomelin (32), 4-dechlorothiomelin (33), 4-dechloro-8-0methylthiomelin (34) and 2-dechloro-8-c?-methylthiomelin (35) were deduced from spectroscopic data (Fig. 15.11). The occurrence of such xanthones in lichens is not entirely unexpected since the probable precursor anthraquinones, such as islandicin (36) and structurally related bis-xanthones, secalonic acid A (37) and secalonic acid C (38), are known to occur in both lichenised and non-lichenised fungi.

15.

OH

Ο

OH

527

L I C H E N SUBSTANCES

Ο

OH

OH

Ο

OH

OH

O-methylation

* TH2,39 FIG. 15.12.

37 38

R 1 = < * - C 0 2M e , R 2 = RT = £ - C 0 2M e , R 2 =

β-Me a-Me

Biosynthesis of xanthones and derivatives.

Rinodina thiomela was also found to contain trace quantities of 2,4-dichloro-lhydroxy-7-methoxy-6,8-dimethyl-9//-xanthone (39), while l,8-dihydroxy-3,6-dimethoxy-9//-xanthone (40) was isolated from a Diploschistes sp. (Elix et al, 1987b). The derivatives (39) and (40) constitute the third and fourth known substitution pattern observed for xanthones from lichens. The latter c o m p o u n d had been previously prepared in a base-catalysed cyclisation of a masked, linear polyketide (41), which, in retrospect, could be considered a biomimetic synthesis. 4.

Depsidones

In 1985 Gunzinger and Tabacchi (1985a) reported the isolation of the novel depsidone, furfuric acid (42), from extracts of the lichen Pseudevernia furfuracea (L.) Zopf. The structure of this c o m p o u n d was deduced from the spectroscopic properties of this Me

OH

M e 0 2C ^ A ^ M e OH

44

Me 9

42

CH 2OAc

O ^ ^ C 0

Me

CHO FIG. 15.13.

45

2

V ^ C 0

Me

CHO

H

2

M e

528

C.

F.

CULBERSON

AND

J. A .

ELIX

compound and was confirmed by total synthesis of the corresponding methyl ester (Gunzinger and Tabacchi, 1985b). However, more recent experiments have shown that (42) is formed in one step by the acid-catalysed alkylation of methyl β-orsellinate (43) or the common depside atranorin (44) with physodalic acid (45) (Elix et al, 1987a) (Fig. 15.13). This mode of synthesis gives credence to the suggestion that (42) is an artifact of the isolation procedure rather than a true metabolite of P. furfuracea. C.

N e w M e t h o d o l o g y for S t r u c t u r a l Elucidation

The application of physical techniques, particularly proton and carbon-13 N M R spectroscopy, mass spectrometry and X-ray crystal analysis, has increased the ease and rate of structural investigations of natural products. M o r e specifically, Sundholm and Huneck (1980, 1981) recently made a detailed study of the carbon-13 spectra of a number of orcinol and β-orcinol depsides and depsidones. In several instances the detailed analysis of N M R data and the development of sophisticated techniques have led to the structural elucidation of new metabolites, e.g. the new depsidones, glomelliferonic acid (46), loxodellonic acid (47) and glomellonic acid (48) (Elix et al, 1987e) (Fig. 15.14). Additional structural information is often forthcoming from double irradiation experiments, 2D-COSY spectra, partially relaxed spectra, and Tx measurements. A carbon-13/proton 2D N M R experiment on the depsidone (46) confirmed the carbon-13 and proton N M R assignments, while by using Tl values even the methylene carbon signals in the carbon-13 spectrum of (46) could be assigned to individual side chain carbon atoms, since the more remote the methylene carbon is from the aromatic ring, the higher the observed 7\ value. However, the real utility of such structural probes is best demonstrated in carbocyclic systems, and they have been used to advantage in the structural elucidation of minute quantities of the new triterpene, aipolic acid (Elix et al, unpubl. res.). X-Ray crystal analysis has also been used in the unambiguous, structural elucidation of several new, atypical, lichen metabolites including thiomelin diacetate (31) (Elix et al., 1987d), eriodermin (50) (Connolly et al., 1984) and wrightiin (51) (Maass and Hanson, 1986) (Fig. 15.14). C 3H 7

46 47 48

R

H 3C 50 FIG. 15.14.

R R R

C 0 2H

Cl 51

15.

LICHEN

Me

FIG. 15.15.

529

SUBSTANCES

Me

Me

Biomimetic-type synthesis of dibenzofurans.

FIG. 15.16.

D. 1.

N e w S y n t h e t i c Routes t o Dibenzofurans and Depsidones Biomimetic-type

syntheses

A synthesis of the diphenyl ether, methyl micareate (27), involving an intramolecular Smiles rearrangement, has been described above (Elix et al., 1987g) (Fig. 15.6). This synthetic methodology has since been elaborated to afford a biomimetic-type synthesis of the lichen dibenzofurans, schizopeltic acid (52) and pannaric acid (53) (Fig. 15.15) (Elix and Parker, 1987). The biosynthetic interrelationship between the c o m m o n lichen depsides and depsi-

530

C.

F.

CULBERSON

AND

depsidones FIG. 15.17.

J. A .

ELIX

diphenyl ethers

Suggested biosynthetic route to depsidones.

dones has been a subject of speculation for some time. Circumstantial evidence exists for such an interrelationship in the form of the co-occurrence of iso-structural depsidedepsidone pairs, such as olivetoric acid (54) and physodic acid (55) in the same organism (Fig. 15.16). In view of this Elix et al. (1987f) suggested that depsidones are derived from />0ra-depsides, as outlined in Fig. 15.17. Thus C-hydroxylation of the 5'-position would be followed by acyl migration and subsequent Smiles rearrangement of the raeta-depside formed, to lead to the corres­ ponding orcinol-depsidones. Although this suggestion needs to be verified by in vivo labelling experiments, the above authors employed such a biomimetic-type approach to synthesise the new depsidones divaronic acid (56) and stenosporonic acid (57), as outlined in Fig. 15.18 (Elix et al, 1987f).

531

15. LICHEN SUBSTANCES C3H7 η

η

P h C H 20

OH

PhCH

I I

1 1

+

R = C3H7, 0 5Η 1Ί (CF 3CO) 20

OH-

(CF 3CO) 20

FIG. 15.18.

2.

56 57

R = C3H7 R = C 5H n

Biomimetic-type synthesis of divaronic and stenosporonic acids.

Partial synthesis of

depsidones

When the prime objective is to effect structural confirmation of a new depsidone, a partial synthesis from a known (natural) depsidone often provides the most effective and efficient means of doing so. The structure of the new depsidones dechloropannarin, isovicanicin and allorhizin (58) (Elix et al, 1982); isonotatic acid and subnotatic acid (Elix and Lajide, 1984); α-acetylconstictic acid (Elix et al, 1987c); a-acetylhypoconstictic acid (Elix et al., 1985); and norpannarin, norargopsin and nordechloropannarin (Elix et al, 1986) have all been verified by such means. The synthesis of allorhizin (58) from natural hypostictic acid (59) is typical and is outlined in Fig. 15.19 (Elix et al., 1982).

532

C.

F. C U L B E R S O N

A N D J. A .

ELIX

Lil HCONMe 2

Ο Me \\

Me

Me

CHO 58

FIG. 15.19.

Partial synthesis of allorhizin from hypostictic acid.

ACKNOWLEDGEMENT This work was supported in part by grant BSR-85-07848 from the National Science Foundation to Duke University.

REFERENCES A r c h e r , A . W . (1978). J. Chromatogr. 152, 2 9 0 - 2 9 2 . A r c h e r , A . W . (1981). Lichenologist 13, 2 5 9 - 2 6 3 . A s a h i n a , Y . (1936). J. Jap. Bot. 12, 8 5 9 - 8 7 2 .

15.

LICHEN

SUBSTANCES

533

A s a h i n a , Y . a n d S h i b a t a , S. (1954). " C h e m i s t r y o f L i c h e n Substances." J a p a n Society f o r t h e P r o m o t i o n o f Science, T o k y o . Ascaso, C . a n d G a l v a n , J . (1976). Pedobiologia 16, 3 2 1 - 3 3 1 . Ascaso, C , G a l v a n , J. a n d O r t e g a , C . (1976). Lichenologist 8, 1 5 1 - 1 7 1 . Baker, J. K . a n d M a , C . - Y . (1979). / . Chromatogr. 169, 107-115. Bendz, G , Santesson, J. a n d W a c h t m e i s t e r , C . A . (1965). Acta Chem. Scand. 19, 1776-1777. Bendz, G , Santesson, J . a n d T i b e l l , L . (1966). Acta Chem. Scand. 2 0 , 1181. 23, 857-858. C o n n o l l y , J. D . , Freer, Α . Α . , K a l b , K . a n d H u n e c k , S. (1984). Phytochemistry C u l b e r s o n , C . F. (1969). " C h e m i c a l a n d B o t a n i c a l G u i d e t o L i c h e n P r o d u c t s . " U n i v e r s i t y o f N o r t h C a r o l i n a Press, C h a p e l H i l l . C u l b e r s o n , C . F. (1970). Bryologist 7 3 , 177-377. 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" S e c o n d S u p p l e m e n t t o ' C h e m i c a l a n d B o t a n i c a l G u i d e t o L i c h e n P r o d u c t s ' " A m e r i c a n B r y o l o g i c a l a n d L i c h e n o l o g i c a l Society, St. L o u i s . C u l b e r s o n , C . F., N a s h , Τ . H . a n d J o h n s o n , A . (1979). Bryologist 8 2 , 1 5 4 - 1 6 1 . C u l b e r s o n , C . F., C u l b e r s o n , W . L . a n d J o h n s o n , A . (1981). Bryologist 8 4 , 16-29. C u l b e r s o n , C . F., C u l b e r s o n , W . L . a n d J o h n s o n , A . (1983). Biochem. Syst. Ecol. 1 1 , 7 7 - 8 4 . C u l b e r s o n , C . F., H a l e , Μ . E., T o n s b e r g , T . a n d J o h n s o n , A . (1984). Mycologia 7 6 , 148-160. C u l b e r s o n , C . F., C u l b e r s o n , W . L . a n d J o h n s o n , A . (1985). Bryologist 8 8 , 3 8 0 - 3 8 7 . C u l b e r s o n , C . F., C u l b e r s o n , W . L . , G o w a n , S. a n d J o h n s o n , A . (1987). Am. J. Bot. 7 4 , 4 0 3 - 4 1 3 C u l b e r s o n , C . 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