Lichen are complex organisms consisting of a symbiotic association of a fungus and an alga. The morphology, physiology and biochemistry of a lichen is different to that of the isolated fungus and alga.^1,2 Lichen have, among other things, developed a number of strategies to minimize UV damage. The synthesis or bioaccumulation of different compounds that directly or indirectly absorb UV energy is one such strategy.^3,4
Previous phytochemical studies of Stereocaulon ramulosum revealed the presence of perlatolic acid, methyl βorcinolcarboxylate and atranorin, from a sample that was collected from Pongo 3800 m.a.s.l. (La Paz, Bolivia) in November. The presence of perlatolic acid was especially interesting, as it shows activity as an antioxidant and a photoprotector of UVB (280-315 nm).⁵ In this paper, we describe a similar study of the same species from the same habitat but collected in June. From this we isolated and determined the structure of 1,3,7-trimethylguanine (1), perlatolic acid (2), methyl β-orcinolcarboxylate (3), atranorin (4) and galactitol (5), see Figure 1 for structures.

Compound 1 was obtained as a white crystals m.p. 238^oC. The elemental composition C₈H₁₁ON₅ was deduced from the EIMS spectrum (m/z 193) and ¹³C NMR spectrum. The IR spectrum exhibited strong absorptions at υ_max3340, 1702 and 1658 cm^-1 indicative, for N-H, C=O, C=N group and an aromatic ring. The ¹H NMR spectrum revealed the presence of three methyl groups at δ 3.39 s, δ 3.57 s, δ 3.98 s and one aromatic proton at δ 7.50 s. The ¹³C NMR spectrum displayed signal for eight carbon atoms, including four non-protonated unsaturated carbons, one protonated and three methyls. The ¹³C NMR data suggest that compound 1 is a guanine derivate. HMBC and COSY correlations observed were in agreement with the guanine skeleton. Important correlations were those observed in the ¹H-¹H COSY experiment and the HMBC (Table 1).

In the HMBC spectrum, δ 3.39 correlate with C-2 (δ 151.9) and C-6 (δ 155.9); δ 3.57 with C-2 (δ 151.9) and C-4 (δ 149.1); δ 3.98 with C-5 (δ 107.6) and C-8 (δ 141.8). The singlet aromatic proton at δ 7.50 correlated with C-6 (δ 155.9), C-5 (δ 107.6), C-4 (δ 149.1) and methyl carbon at δ 34.0 (Figure 2).

From the above evidence, compound 1 was characterized as 1,3,7-trimethylguanine (Figure 3).

The same compound was previously found in different species of sponges,^6,7,8,9 although this is the first time it is reported from a lichen.

The structures of perlatolic acid, atranorin, methyl β-orcinolcarboxylate and galactitol were elucidated by spectroscopy of ¹H NMR, ¹³C NMR, COSY, HMQC, HMBC and by comparison of their spectroscopy data with those reported in the literature.^10,11,12,13

General

All melting points were recorded with a Reichter microscope. The UV and IR spectra were recorded with a Varian Cary 2290 and a Perkin-Elmer 298 spectrometer, respectively. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded in CDCL₃ and MeOD usin a Bruker DRX400 spectrometer with an inverse multinuclear 5-mm probe head equipped with a shielded gradient coil. COSY, HMQC and HMBC experiments were recorded with gradient enhancements using-sine-shape gradient pulses. EIMS were recorded with a JEOL SX102 spectrometer at 70 eV. Column chromathography was\run on Merk silica gel 60 and TLC was carried out on Silica gel GF₂₅₄.

Plant Material

The lichen Stereocaulon ramulosum Rausch were collected from Pongo-La Paz Bolivia at 3800 m.a.s.l. in June 2002. Voucher specimens (JV-001) were deposited at the Herbarium National of Bolivia.

Stereocaulon ramulosum (254.0 g) were extracted successively with petroleum ether, CH₂Cl₂, Me₂CO and EtOH, to yield a crude organic extract (14.6 g) on evaporation in vacuo. This extract was subjected to CC on silica gel eluted with CH₂Cl₂-Me₂CO mixtures of increasing polarity to afford guanine 1 (40 mg), perlatolic acid (2) (250 mg), methyl β-orcinolcarboxilate (3) (65 mg), atranorin (4) (800 mg), and galactitol (5)(32 mg).

1,3,7-trimethylguanine (1); white crystals (MeOH-(CH3)₂CO); m.p. 238^oC; IR (KBr) υ_max 3340, 1702, 1658 cm^-1; ¹H and ¹³C NMR, see Table 1. EIMS (70 e/v) m/z [M]⁺ 193 (100), 164 (8), 136 (5), 137(5), 109 (30), 82 (10), 67 (10), 55 (10) (C₈H₁₁ON₅).

Perlatolic acid (2); white crystals (MeOH-(CH₃)₂CO); m.p. 107-108^oC; ¹H NMR (CDCl₃,400 MHz); δ 0.85 (CH₃ε), 0.9 (CH₃-ε’), 1.2-1.5 (CH₂-γγ’,δδ’), 1.6-1.8 (CH₂-β,β’), 2.9 (CH₂-α), 3.0 (CH₂-α’), 3.8 (OCH₃), 6.39 (CH₃), 6.65 (CH-5’), 6.75 (CH3’); ¹³C NMR (CDCl₃, 100 MHz); δ 13.9, 13.9, 22.4, 22.6, 31.3, 31.6, 32.0, 36.4, 37.2, 55.4, 99.0, 103.5, 108.6, 109.5, 111.9, 116.1, 148.4, 150.1, 155.0, 164.9, 165.2, 166.5, 169.4, 175.5; EIMS (70 e/v) m/z [M]⁺ 444 (2), 386 (19), 368 (24), 277 (35), 256 (40), 221 (69), 180 (31), 164 (84), 131 (81), 124 (100), 107 (64) (C₂₅O₇H₃₀).