Phycomysterols and Other Sterols from the Fungus Phycomyces blakesleeanus

Abstract

In the search for novel bioactive products from filamentous fungi, sterols and triterpenoids found in Phycomyces blakesleeanus were analyzed using semipreparative HPLC, GC-MS, and NMR techniques. Structures proposed for the three new compounds identified, phycomysterol A (1), phycomysterol B (2), and neoergosterol (3), were confirmed by chemical synthesis. Phycomysterols possess a new natural 19-norergostane skeleton with an aromatic B ring. Phycomysterol A showed anti-HIV activity.Fungi have been one of the main natural sources of biologically active substances since the discovery of antibiotics. Searching for new bioactive products from filamentous fungi, chemical analysis and biological screening of metabolites from Gibberella fujikuroi have been the subject of our work over the past few years. [1][2][3] We recently began studying Phycomyces blakesleeanus (Mucoraceae), which is a fungal producer of -carotene (provitamin A), riboflavine (vitamin B 2 ), pyridoxine (vitamin B 6 ), ergosterol (provitamin D 2 ), biotin, and nicotinic acid. 4,5 Therefore, oil from Phycomyces could be an interesting alimentary vitamin complement, if it were proven to be free of mycotoxins. We have identified more than forty nontoxic compounds among the acidic metabolites from P. blakesleeanus 6 and are now working on neutral metabolites. In the mycelium of P. blakesleeanus (NRRL1555 wild strain) we have found eleven sterols previously unreported in Phycomyces. These include the new natural sterols (1-3; Chart 1) with a 19-norergostane skeleton and an aromatic B ring. Previous studies of sterols from Phycomyces at the University College of Wales yielded a number of new sterols (4-13) and certain aspects of fungal ergosterol biosynthesis were resolved. 7-11 However, some sterols present in the fungus were not identified. 9 Results and DiscussionSterol and triterpene mixtures isolated from P. blakesleeanus were subjected to semipreparative HPLC. Fractions obtained were studied by NMR spectroscopy and aliquots were treated with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to form TMS derivatives for analysis by GC-MS. The sterol ester mixture was saponified, and the nonsaponifiable fraction was analyzed as described above. Results are shown in Table 1. Proportions are only approximate because they were determined from the area of the gas chromatogram peaks, based on total ion current response data. TMS-ethers of sterols were used for GC-MS analysis, instead of free sterols, because the former have better chromatographic properties and afford highly characteristic modes of fragmentation from which structural details may be inferred. 12 In the mass spectrum of 1, as the TMS-ether, the molecular ion was consistent with the TMS derivative of a sterol with the molecular formula C 27 H 40 O, containing four double bonds. The fragment ions [M -TMSOH -C 9 H 17 ] + , [M -TMSOH -C 6 H 11 ] + , and [M -(C 6 H 11 + H)] + indicated a side chain with nine carbon atoms and a double bond. Thus, 1 must possess a nucleus with three double bonds and ...

Sterols in erg mutants of Phycomyces: metabolic pathways and physiological effects

“…The fact that 22 was produced at all in mutant strains is the first experimental evidence demonstrating that the sterol C-22 dehydrogenase of Phycomyces is capable of recognizing sterols with the 24(28) unsaturated side chain, supporting the hypothesis that both routes may be taken in wild-type Phycomyces. On the basis of these results and the chemical structures of sterols and triterpenoids previously found in the wild-type strain NRRL1555 [11], a biosynthetic path- way for sterols in Phycomyces is proposed in Scheme 1. The presence of 22 in the mutants, but not of its counterpart 23 in the wild-type, can be explained by a slower rate of conversion of 22314 (relative to the rate of conversion of 2339 in the wild-type) and hence its accumulation.…”

“…Media were solidified with 1.5% (plates) or 0.7% (top agar) agar. For experiments on sterol production, mutant strains H23, H25, H37 and H38 were incubated in 50 flasks, each containing 100 ml of minimal medium, as previously described for wild-type Phycomyces [11]. Additionally, the media for mutant H37 contained nicotinic acid (for which it is auxotrophic) to a final concentration of 1 mg/l (sterilized by filtration through a 0.22 m-pore membrane, Millipore).…”

“…The dry weights of mycelia obtained for each strain were: H23, 27.70 g; H25, 36.35 g; H37, 9.80 g; and H38, 38.39 g. Sterols were extracted and analyzed by means of semipreparative HPLC, NMR spectroscopy and GC-MS techniques, as previously reported for the wild-type strain [11]. Mass spectra and relative retention times of trimethylsilyl (TMS) ethers of 1-21 matched those already reported [11]. Ergosta-7,22,24(28)-trien-3␤-ol (22) (8).…”

“…The results are shown in Table 1. Sterols and triterpenoids previously found in the wild type strain (1-21) [11] have been also included in Table 1, in order to facilitate further comparison with mutants. Sterol 22, found in mutants, was not previously detected in the wild type strain.…”

“…However, in spite of their presumable usefulness for research on sterol biosynthesis, erg mutants of Phycomyces have not been previously reported. Recently, an exhaustive analysis of sterols and triterpenoids from Phycomyces was carried out in our laboratory, and thus more than twenty compounds were unambiguously identified in the wild-type strain NRRL 1555 [11]. With the sterol profile of the wild-type fungus well established, we have isolated erg mutants of Phycomyces and submitted them to chemical and physiological analysis.…”