Lichenologist 34(0): 000–000 (2002)

Halogenated anthraquinones from the rare southern Illinois lichen Lasallia papulosa

Peter A. COHEN

Abstract: Four anthraquinones were isolated from the foliose lichen, Lasallia papulosa (Ach.) Llano. Two of the anthraquinones are known compounds, previously isolated from Lasallia papulosa, while the other two were reported previously as secondary metabolites from laboratory-cultured Nephroma laevigatum, and are isolated here for the first time from lichens in their natural habitat. All compounds were characterized by UV spectrophotometry, mass spectrometry, 1H NMR and 13C NMR. The products were identified as 7-chloroemodin, valsarin (7-chloro-5-hydroxyemodin), 5-chloro-1-Omethylemodin and 5-chloro-1-O-methyl--hydroxyemodin. 2002 The British Lichen Society

Key words:: ??

Introduction

In our previous examination of pigmentcontaining foliose lichens, we found several genera of temperate lichens containing chlorinated anthraquinones (Cohen & Towers 1995a, 1995b, 1997). In this chemical group halogenated anthraquinones bearing a chlorine atom in the 7-position of the ring predominate over structural analogues with chlorine in the 5-position (Huneck & Yoshimura 1996). This substitution pattern probably reflects the electronic character of the anthraquinone ring and is consistent with known in vitro mechanisms of electrophilic chlorination with halogens (Lam et al. 1972). However, 5-chloroanthraquinones have been previously isolated from Nephroma laevigatum when grown in a culture supplemented with radioactive chlorine (Cohen & Towers 1996), although they have not heretofore been identified in natural populations of lichens (Huneck & Yoshimura 1996). The compound 5-chloro-1-O-methylemodin has

been previously isolated from a nonlichenized ascomycete (Ayer &

Trifonov 1994), but has not been reported previously from Lasallia papulosa or any other lichen (Posner et al. 1990; Posner et al. 1991).

We report here the isolation of two known naturally-occurring lichen substances, 7-chloroemodin (1) and valsarin (2), and two other secondary metabolites, 5-chloro1-O-methylemodin (3) and 5-chloro-1-Omethyl--hydroxyemodin (4), known previously only from laboratory-cultured Nephroma laevigatum (Cohen & Towers1996).

The disjunctive global distribution of Lasallia papulosa has been described previously

doi:10.1006/lich.2002.0419, available online at http://www.idealibrary.com on

(Llano 1950). It is rather abundant in the Appalachian mountains of eastern North America, uncommon in the western Rocky Mountains and Black Hills region, and rare in southern Illinois where it

was collected for this present study (Brodo et al. 2001). In this region there is only one published report from a single location on the Cretaceous sandstone outcrop known as the

P. A. Cohen: Department of Biology, Adelphi University, Garden City, New York 11530, U.S.A.

‘Garden of the Gods’ in southern Illinois (Skorepa 1973). This lichen is often referred to as ‘toadskin lichen’ because of its brown0024–2829/02/000000+00 $35.00/0 2002 Published by Elsevier Science Ltd on behalf of The British Lichen Society.

2 THE LICHENOLOGIST Vol. 33

Table 1. HPLC data for compounds isolated from Lasallia papulosa*

Compound* Retention time† (min)

7-Chloroemodin (1) 4·23

Valsarin (2) 3·96

5-Chloro-1-O-methylemodin (3) 3·45

5-Chloro-1-O-methyl--hydroxyemodin (4) 3·23

*All compounds were eluted from a reversed-phase Waters Bondapak C18 column (440 cm; 10 M) with a flow rate of 1 ml min1.

†Solvent system: gradient of 1:1 MeOH-2% AcOH in CH3CN to 100% MeOH in

15 min.

exterior and warty surface texture (Goward 1996). In general, the lichen is brown on the exterior, and has a brown to black underside; however, there are species that are bright red on the surface and brown-red on the underside. No highly pigmented forms of Lasallia papulosa were found in ‘Garden of the Gods’, although they have been reported from West Virginia (Brodo et al. 2001). Highly pigmented varieties [L. papulosa var. rubiginosa (Pers.) Llano] have been collected from Table Mountain near Cape Town, South Africa (Llano 1950).

Materials and MethodsGeneral

Melting points: uncorrected; EIMS; 70 eV; 1H NMR: 400 MHz; 13C NMR 125 MHz; DMSO-d6, DMF-d6 and Me2CO-d6 were used in the NMR and NOE experiments (TMS as internal standard);

Column Chromatography (CC): BDH flash silica gel 60 (230– 400 mesh); preparative TLC: Merck Kieselgel 60 GF254 layers (0·22020 cm) on glass plates; HPLC: Waters reversed-phase C18 column (440 cm, gradient elution, 10 m, flow rate 1 ml min1) (seeTable 1); UV spectra: MeOH (UV grade); CHCl3

and CH2Cl2 (solvent grade).

Lichen material

Lasallia papulosa (Ach.) Llano was collected from two north-facing red sandstone outcrops in the park, ‘Garden of the Gods’, Saline County, southern Illinois, on 11 November 2000. In a recent survey of this region, two distinct lichen locations were identified, in a region covering approximately five square miles. The samples obtained for this present study came from both locations. One reference sample has been deposited in the Herbarium in the Department of Plant Biology at Southern Illinois University in Carbondale (Herbarium Number: 42016).

Isolation and identification

The carefully cleaned lichen (100 g dry weight) was immersed in 500 ml 95% MeOH for 48 h, without damage to the lichen tissue. The red-brown extract was concentrated in vacuo yielding a red waxy solid. This solid

was freeze-dried to give a red-brown powder (15 g). This powder was purified by CC on Sephadex LH-20, using a gradient of CH2Cl2-MeOH (9:1) to MeOH. Fractions (40 ml) were collected and analysed by TLC and UV light (short-wave and long-wave). The fractions containing anthraquinones were further purified by CC on Sephadex LH-20, using a gradient of CH2Cl2-MeOH (6:4) to MeOH. Fractions (20 ml) were collected and analysed by TLC and UV light. All compounds were further purified by preparative TLC (CH2Cl2-MeOH 4:1) and repeatedly crystallized until purity was established to be >99% by RP-HPLC. All compounds were characterized by UV, MS, and 1H NMR spectra (in addition, compounds 2 and 3 was characterized by 13C NMR spectra).

Preparation of acetylated derivatives

The anthraquinone (2 mg) was suspended in dry pyridine (1 ml) to which was added 0·5 ml of dry acetic anhydride. The solutions were left at room temperature over night, and then concentrated in vacuo to a solid which was vacuum dried for 48 h. The acetylated derivatives were purified by preparative TLC, and recrystallized from EtOAc.

Results

The extraction of approximately 100 g (dry weight) of lichen thalli in MeOH produced a brown-red solution, which upon TLC development (9:1 CHCl3-MeOH) yielded four (pigmented) primary spots, and three minor (non-pigmented) spots. The non-pigmented spots included the known compounds gyrophoric acid (Rf 0·2) and umbilicaric acid

(Rf 0·3), well-described lichen depsides AOH O OH

R O

R

1 H 7-Chloroemodin

2 OH Valsarin (7-Chloro-5-hydroxyemodin)

B OH O OCH3

Cl O

R

3 CH3 5-Chloro-1-O-methylemodin

4 CH2OH 5-Chloro-1-O-methyl- -hydroxyemodin ω

F. 1. Anthraquinone isolated from Lasallia papulosa.A, 7-Chloroanthraquinones; B; 5-

Chloroanthraquinones.

(Culberson 19697). The pigmented spots consisted of: 1 (Rf 0·5), 2 (Rf 0·4), 3 (Rf 0·65) and 4 (Rf 0·15). The structures of compounds 1, 2, 3 and 4 are shown in Fig. 1

The identity of 1 was established by 1H NMR and mass spectra. The spectral data are consistent with previous results (Cohen& Towers 1995a, b, 1997; Huneck & Yoshimura 1996). Compounds 2, 3 and 4were confirmed by 1H NMR (2, 3 and 4), 13

C NMR (2 and 3) and mass spectra (2, 3 and 4). Spectral data, in particular NOE experiments, were consistent with previous results for 3 obtained from cultured N. laevigatum (Cohen & Towers 1996). Thus,

when 3 was submitted to irradiation at the H-2 proton (see Fig. 1), a concomitant enhancement (2%) of the methoxy proton at C-1 was clearly observed in the spectrum. In addition, the 13C NMR spectrum of 3 was indicative of a structure bearing an electronegative atom (Cl) at C-5 consistent with our previous findings (Cohen & Towers 1996). The 1H NMR spectra are indeed distinct for 1 and 3 (and 4), as can be clearly seen in the spectra for 3 and 4, where the characteristic H-7 proton shift ( 6·68) is clearly present indicating that the position between the two hydroxyl groups at C-6 and C-8 bears a proton. This proton is naturally lacking in all 7-chlorosubstituted anthraquinones. The NOE enhancements also

6

7

5

8

10

9a9

a8 a10

a44

3

2

1Cl

3CHHO

RHO

2002 Author please supply short running head—Cohen 3

indicate that there is no chlorine substitution at C-2 (Cohen & Towers 1996).

The identity of 2 was established by 1H NMR, 13C NMR and mass spectra. We report here for the first time the 13C NMR spectrum for valsarin (2). All previous reported structural assignments for this compound were based on data derived from UV and IR spectra, mass spectra, 1H NMR spectra and elemental analysis (Huneck & Yoshimura 1996; Lam et al. 1972; Posner et al. 1990; Posner et al. 1991).

The basis for establishing the presence of a -hydroxy functionality in 4 was the distinctive splitting pattern (and coupling constant) in the proton spectrum. The methylene protons adjacent to the OH group ( 4·64) produced a d with a J=5·0 Hz, the same coupling constant observed in our earlier work (Cohen & Towers 1996).

The Compounds

7-Chloroemodin (1). Compound 1(12 mg, 0·12% dry weight) was obtained as orange crystals (EtOAc); mp 282–2840. The 1 13

H and C NMR spectra were in agreement with the literature (Huneck & Yoshimura 1996).

7-Chloro-5-hydroxyemodin (valsarin) (2). Compound 2 (23 mg, 0·23% dry weight) was obtained as red crystals (EtOAc); mp 268–2700; UV (MeOH) max

nm (log ) 238 (4·25), 264 (4·08), 312 (3·89), 350 (3·64), 468 (3·82), 492 (3·55), 520 (3·20), 574 (3·18); EIMS (70 eV) m/z (rel. int.) 322 [M]+ (30) 320

(100); 1H NMR (400 MHz, DMSO-d6): 2·38 (3H, s, Me-3), 7·12 (1H, s, H-2), 7·52 (1H, s, H-4);13

C NMR (125 MHz, DMSO-d6): 21·8(Me-3), 108·9 (C-9a), 110·2 (C-8a), 120·3

(C-4), 122·3 (C-2), 126·4 (C-7), 133·0 (C10a), 133·4 (C-4a), 144·3 (C-5), 147·6 (C3), 156·3 (C-6), 158·2 (C-8), 159·4 (C-1), 183·4 (C-10), 190·3 (C-9).

7-Chloro-5-hydroxyemodin tetraacetate (tetraacetylvalsarin). Yellow-orange crystals from EtOAc; mp 220–2220; CIMS m/z (rel. int.) 491 [MH]+

(32), 489 (100); EIMS (70 eV) m/z (rel. int.) 488 [M]+ (8),446 (10), 436 (23), 404 (38), 362 (52), 322 (22), 320 (100); 1H NMR (400 MHz, Me2CO-d6): 2·38 (3H, s, Me-3), 2·422·48 (12H, s, 1,5,6 and 8-OAc), 7·18 (1H, s, H-2), 7·78 (1H, s, H-4).

5-Chloro-1-O-methylemodin (3). Compound 3 (8 mg, 0·08% dry weight) was obtained as orange crystals (EtOAc); mp 252–2540; UV (MeOH) max nm (log ) 230(4·40), 260 (4·34), 316 (4·24), 440 (3·92); EIMS (70 eV) m/z (rel. int.) 320 [M+] (33) 318 (100); 1H NMR (400 MHz, DMSOd6): 2·48 (3H, s, Me-3), 3·94 (3H, s, OMe-1), 6·78 (1H, s, H-7), 7·36 (1H, s,

H-2), 7·52 (1H, s, H-4); 13C NMR(125 MHz, DMSO-d6): 22·0 (Me-3), 57·5 (OMe-1), 112·8 (C-7), 113·0 (C-8a), 113·6 (C-9a), 118·6 (C-5), 120·0 (C-4), 121·2(C-2), 130·4 (C-4a), 135·3 (C-10a), 146·2 (C-3), 160·0 (C-1), 160·4 (C-8), 162·0 (C-6), 184·3 (C-10), 192·3 (C-9).

5-Chloro-1-O-methyl--hydroxyemodin (4). Compound 4 (4 mg, 0·04% dry

weight) was obtained as orange-red crystals (AcOH); mp 264–2660; UV (MeOH) max nm (log ) 220 (4·50), 256 (4·20), 322 (3·98), 498 (3·45); EIMS (70 eV) m/z (rel. int.) 336 [M+] (35) 334 (100); 1H NMR (400 MHz, DMSO-d6): 3·92 (3H, s, OMe-1), 4·64 (2H, d, J=5·0

4 THE LICHENOLOGIST Vol. 33

Hz, CH2OH3), 6·0 (1H, s, CH2OH-3), 6·78 (1H, s, H-7), 7·48 (1H, s, H-2), 7·78 (1H, s, H-4).

5-Chloro-1-O-methyl--hydroxyemodin triacetate. Orange crystals from EtOAc; mp 244–2460; CIMS m/z (rel. int.) 463 [MH]+ (33), 461 (100); EIMS (70 eV) m/z

(rel. int.) 462 [M]+ (7), 420 (22), 418 (44), 378 (20), 376 (60), 336 (4), 334 (8), 318 (38), 316 (100), 306 (12), 304 (40).

Discussion

Compound 3 had originally been isolated from the non-lichenized ascomycete fungus Phialophora alba (Ayer & Trifonov 1994), but this is the first reported isolation of a 5-chloroanthraquinone from a lichen collected in the field. Previously, compound 4 was only known from axenically cultured Nephroma laevigatum. Since 5-chloroanthraquinones are relatively infrequent in both lichenized and non-lichenized ascomycetes relative to the 7-chloro analogues, one might ponder on the biochemical mechanisms for chlorination. Since the 7 position is more nucleophilic than the 5 position in the anthraquinone ring (Lam et al. 1972), the evidence suggests that a chloroperoxidase capable of selectively chlorinating the less-nucleophilic C-5 position must be present in L. papulosa. We have previously demonstrated, in vivo, that axenically cultured lichen-forming fungi have the capacity to biosynthesize novel analogues through (presumably) physiological and biochemical perturbation of their polyketide pathways that produce anthraquinones (Cohen & Towers 1996). Subsequently, we have shown that in vitro chlorination of anthraquinones with a partially purified chloroperoxidase isolated from Nephroma laevigatum, as well as by commercial fungal chloroperoxidasecatalysed chlorination of anthraquinones (Cohen & Towers 1997). Regioselectivity was clearly observed in this

experiment, as the enzyme from the non-lichen-forming fungus was capable of generating5-chloroanthraquinones, while the lichen enzyme strictly catalysed introduction of chlorine into the C-7 position. It therefore remains to be established whether the unique geological environment where L. papulosa was

found might have some bearing on the ability of this lichen to biosynthesize rare chlorinated isomers of well-known lichen pigments. Future research will be directed toward studying the in vitro and in vivo biochemical mechanisms for chlorination in Lasallia, utilizing whole organism cultures, purified enzymes and labelled precursors.

Financial support was provided by the Department of Chemistry and Biochemistry at Southern Illinois University in Carbondale. I am most grateful to Professor Cal Y. Meyers for the use of his laboratory (Meyers Institute), to Mr Paul Sandrock for his kind assistance with the proton and carbon spectra, and Dr Steve Mullen of the Mass Spectrometry Laboratory at the University of Illinois Urbana-Champaign for running mass spectra.

RAyer, W. A. & Trifonov, L. S. (1994) Anthraquinones and

a 10-hydroxyanthrone from Phialaphora alba. Journal of Natural Products 57: 317–319.

Brodo, I. M., Sharnoff, S. D. & Sharnoff, S. (2001) Lichens of North America. New Haven: YaleUniversity Press.

Cohen, P. A. & Towers, G. H. N. (1995a) Anthraquinones and phenanthroperylenequinones from Nephroma laevigatum. Journal of Natural Products 58: 520–526.

Cohen, P. A. & Towers, G. H. N. (1995b)The anthraquinones of Heterodermia obscurata. Phytochemistry 40: 911–915.

Cohen, P. A. & Towers, G. H. N. (1996) Biosynthetic studies on chlorinated anthraquinones in the lichen Nephroma laevigatum. Phytochemistry 42: 1325– 1329.

Cohen, P. A. & Towers, G. H. N. (1997) Chlorination of anthraquinones by lichen and fungal enzymes. Phytochemistry 44: 271–274.

Culberson, C. F. (1969) Chemical and Botanical Guide to Lichen Products. Koenigstein: Koeltz.

Goward, T., McCune, B. & Meidinger, D. (1994)Lichens of British Columbia. Illustrated Keys: Part I, Foliose and Squamulose Species. Victoria: Ministry of Forests.

2002 Author please supply short running head—Cohen 5

Huneck, S. & Yoshimura, I. (1996) Identification of Lichen Substances. Berlin: Springer.

Lam, J. K. K., Sargent, M. V., Elix, J. A. & Smith, D. O’N. (1972) Synthesis of valsarin and 5,7dichloroemodin. Journal of the Chemical Society, Perkin I: 1466–1470.

Llano, G. A. (1950) A Monograph of the Lichen Family Umbilicariaceae in the Western Hemisphere. Washington, DC: Office of Naval Research.

Posner, B., Feige, G. B. & Huneck, S. (1990) Phytochemische untersuchungen an

westeuropäischen Lasallia- arten. Zeitschrift für Naturforschung 45c: 161–165.

Posner, B., Feige, G. B. & Leuckert, C. (1991) Beiträge zur chemie der flechtengattung Lasallia Mérat. Zeitschrift für Naturforschung 46c: 19–27.

Skorepa, A. C. (1973) Taxonomic and Ecological Studies on the Lichens of Southern Illinois. Doctoral Dissertation, Department of Botany, University of Tennessee.

Wetmore, C. M. (1960) A monograph of the lichen genus Nephroma. Publications of the Museum, Michigan State University, Biol. Ser. 1: 369–506.

Accepted for publication 6 September 2002