Austrogracilin A and B, two 2-naphthoic acid derivatives from the mushroom Austroboletus gracilis

From air-dried fruit bodies of Austroboletus gracilis two unique naphthalene derivatives were isolated and their structures elucidated by spectroscopic and synthetic methods. The chemotaxonomic importance of these compounds is discussed.


Introduction
Austroboletus gracilis (Peck) Wolfe, 1 the Graceful Bolete, is a beautiful mushroom which belongs to a remarkable group of fungi with disjunct occurrence in Eastern North America and East Asia. 2 It is widely distributed from Canada to Costa Rica and has also been recorded from Papua New Guinea, Japan, Taiwan, and China.In a preliminary investigation of this mushroom we have shown that badione A is responsible for the reddish-brown colour of its cap. 3 In this communication we report on the isolation of two novel 2-naphthoic acid derivatives from this species.

Results and Discussion
Investigation of the methanol extract from air-dried fruit bodies of A. gracilis by HPLC with diode array-detection revealed the presence of two major constituents with very similar UV spectra.The two compounds, named austrogracilin A (1) and B (3), could be easily obtained pure by preparative HPLC on reversed phase.
Austrogracilin A is a colourless solid, which becomes yellowish on standing in air.Its UV spectrum (MeOH) exhibits absorption maxima at 229, 266, and 307°nm, and in the IR spectrum intensive bands at 3392°(OH), 1686°(C=O), and 1243°(OH) cm −1 are visible.The high resolution mass spectrum of austrogracilin A shows a molecular peak at m/z° 220.0369 corresponding to the molecular formula C 11 H 8 O 5 .
The 1 H NMR spectrum (CD 3 OD) of austrogracilin A displays only three singlets at δ H 7.19, 7.52 (br), and 7.88 (br) and a doublet at δ H 7.20 (J = 1.4 Hz).According to the 1 H, 1 H-COSY spectrum, the doublet arises by coupling with the proton responsible for the broadened signal at δ H 7.88, thus indicating a meta-relationship.In addition, long-range couplings are observed which explain the broadening of the singlets at δ H 7.52 and 7.88.
The 13 C NMR spectrum of austrogracilin A exhibits 11 signals in the range between 100 and 175 ppm, in agreement with the molecular formula.In addition to signals for four aromatic CHgroups at δ C 106.2, 106.3, 112.2, and 122.5, three quaternary aromatic C-atoms at δ C 125.0, 127.2, and 131.5 are visible.Further signals at δ C 149.0, 149.5, and 153.6 point to aromatic carbons carrying OH groups, and the signal at δ C 171.4 can be assigned to a carboxyl group.HSQC and HMBC experiments allowed the assignment of all 1 H-and 13 C-signals, and from the 1 H, 13 C-correlations illustrated in Figure 1 the structure of a 4,6,7-trihydroxy-2-naphthoic acid (1)   can be proposed for austrogracilin A. It is supported by strong NOE correlations between the two peri-protons at δ H 7.19 and 7.88 in the NOESY spectrum.4-Hydroxy-6,7-dimethoxy-2-naphthoic acid (2) is a known compound that can be prepared in three steps from veratraldehyde. 4Demethylation of 2 with BBr 3 yielded 4,6,7-trihydroxy-2naphthoic acid, which was identical with the natural product.
The UV and IR spectra of austrogracilin A and B indicate a close structural similarity of both metabolites.The UV spectrum (MeOH) of austrogracilin B exhibits absorption maxima at 221, 261, and 323°nm, and in the IR spectrum strong bands at 3458 and 3233° (OH), 1687 (CO), and 1254°(OH) cm  Methylation of austrogracilin B (3) with diazomethane yielded the known permethyl derivative 4. The latter has recently been obtained via an intriguing dimerization followed by oxidative cleavage of methyl 3,4-dimethoxycinnamate. 5 The spectroscopic data from naturally derived 4 agree very well with those reported for the synthetic material.

Conclusions
The occurrence of the unique naphthalene derivatives 1 and 3 in Austroboletus gracilis underlines the special taxonomic position of this genus within the Boletineae.Before considering these compounds as chemotaxonomic markers, further species of Austroboletus have to be studied.In a recent phylogenetic investigation based on DNA analysis, Bresinsky and Binder 6 found that Austroboletus gracilis, A. novae-zelandiae, and A. niveus form a genetically well defined group between Leccinum and Tylopilus.In contrast, Austroboletus betula (Schwein.)E. Horak, a North American species with yellow colours, belongs to a different clade including several Boletellus species. 6Therefore, the shift of A. betula from Boletellus to Austroboletus because of spore ornamentation 1b appears not to be justified and should be reversed.This is also supported by the occurrence of variegatic acid, xerocomic acid, and atromentic acid in A. betula, pigments characteristic for the genus Boletellus. 3,7According to a preliminary investigation, 8 most of the variegatic acid is present as a water-soluble sugar conjugate of undetermined constitution which is characterized by a (M − H) − ion at m/z 1181 in the negative ion FAB MS 9 suggesting the presence of five hexose residues.
Fungal material.A. gracilis was collected in autumn 1994, 1996, 1997 in mixed hemlockrhododendron forests near Highlands, N.C., U.S.A.The fruit bodies were air-dried and stored at room temperature.Voucher samples are kept in the herbarium of N. Arnold, IPB, Halle/Saale.Extraction and isolation procedure.The air-dried fruit bodies of A. gracilis (10 g) were pulverized, defatted with petroleum ether and then exhaustively extracted with degassed MeOH under an argon atmosphere in an ultrasound bath.The combined extracts were concentrated under reduced pressure and the residue distributed between water and EtOAc.The water phase was subsequently extracted with 2-butanol, and both organic phases were dried (Na 2 SO 4 ) and concentrated in vacuo.After solid-phase extraction of the combined residues on a RP-18 cartouche, the compounds were separated by preparative HPLC (system 3).The resulting fractions were lyophilized to yield austrogracilin A (1) (14 mg, 0.14% of dry-weight) and austrogracilin B (3) (15 mg, 0.15% of dry-weight).
−1 are visible.High resolution mass measurement of the (M − H) − peak at m/z 247 in the (−)-ESI MS led to the molecular formula C 12 H 8 O 6 for austrogracilin B. The 1 H NMR spectrum (CD 3 OD) of austrogracilin B contains signals of four aromatic protons.Two of the signals appear as broadened singlets at δ H 8.43 und 8.52 and one as doublet at δ H 8.60 (J = 1.3

Issue in Honor of Prof. Rodney Rickards ARKIVOC 2004 (x) 13-19 ISSN 1424-6376 Page 15
The fourth aromatic proton gives rise to a sharp singlet at δ H 7.33.In DMSO-d 6 additional signals for two phenolic protons at δ H 9.82 and 10.29 are visible as well as a broad signal at δ H 12.92, which corresponds to two carboxyl protons.The 1 H, 1 H-COSY spectrum indicates a strong correlation between the protons at δ H 8.52 and 8.60 as well as long-range coupling between the protons at δ H 8.43 and 8.52.The13C NMR spectrum of austrogracilin B contains 12 signals.According to the DEPT NMR spectrum, the signals at δ C 109.9, 112.8, 129.0, and 135.0 can be assigned to aromatic CH-groups, whereas the signals at δ C 125.5, 127.1, 131.3, 131.7 belong to quaternary aromatic carbons.Two additional signals at δ C 148.8 and 152.0 can be assigned to aromatic carbon atoms carrying OH groups, and signals at δ C 170.1 and 171.2 point to the presence of two carboxyl groups.HSQC and HMBC experiments (Figure 2) allowed the assignment of all signals and established structure 3 for austrogracilin B. Like austrogracilin A, 3 shows a strong NOE effect between the two peri-protons in the NOESY spectrum.