New azasteroidal antifungal antibiotics from Geotrichum flavo-brunneum. II. Isolation and characterization.

Abstract

A novel group of antibiotics, comprising microbiologically-active structurallyrelated factors A25822A, B, D, H, L, M and N, produced by culturing Geotrichum flavo-brunneum NRRL 3862 under submerged aerobic fermentation conditions was isolated by extraction. The individual factors were separated and purified by chromatography and crystallization. The major factor, A25822B, a 15-aza-24-methylene-D-homocholestadiene is a white crystalline compound, C28H45NO. The antibiotics are highly active against fungi and marginally active against bacteria.A mixture of broad spectrum antifungal antibiotics was detected in the crude fermentation broth of the fungi Geotrichum flavo-brunneum NRRL 3862. The novel antibiotic complex was isolated by extraction of the whole fermentation broth or mycelium, and the structurallyrelated factors A25822A1), B, D, H, L, M and N were separated and purified by chromatographic techniques and fractional crystallization. The complex, consisting of one major and six minor components, was highly active against a broad spectrum of pathogenic fungi, including Candida species and Trichophyton species.A25822B, biologically the most active component, was inhibitory in a range of <0.0312 -5.0 mcg/ml. The complex was also active against several gram-positive and gram-negative bacteria". IsolationGeotrichum flavo-brunneum was grown under submerged aerobic fermentation conditions on a casein corn steep agar medium for 7 days. Approximately 10 °o of the antibiotic synthesized was released into the fermentation broth while the remaining 90 % was retained by the cells2).The fermentation broth was filtered and the active principles extracted from the cells by multiple methanol extractions. The extracts were combined, concentrated to an aqueous phase and extracted with ethyl acetate at pH 10. The ethyl acetate extract was concentrated to an oily residue. The crude antibiotic complex was a brown, viscous oil which was soluble in most organic solvents, insoluble in water, and stable in solutions over a pH range of at least pH 3 to pH 10, at room temperature. The crude oil was dissolved in diethyl ether and the active compounds extracted with 0.2 N sulfuric acid solutions. The active components were re-extracted from the aqueous phase at pH 10 with n-hexane and ethyl acetate. A25822B, the most abundant of the factors, was recovered from the n-hexane extract by

Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans

“…Both compounds also decreased the unsaturated-to-saturated fatty acid ratio from 2.3 to 1.4. The azasterol A25822B, at 1 jiM, inhibited ergosterol biosynthesis, consistent with its reported effect on A14 reductase (12,25,54). It also decreased the unsaturatedto-saturated fatty acid ratio from 2.3 to 1.3.…”

Structure‐function activity of azasterols and nitrogen‐containing steroids

“…Recently, a novel group of antibiotics produced by culturing Geotrichum flavo-brunneum was isolated. The major active fraction was a 15-a z a-2 4-me t hylen e-D-h omocholestadiene (29). The homoaza steroid was most active against pathogenic fungi, including Candida and Trichophyton species (30).…”

Mode of Action of the Azasteroid Antibiotic 15-Aza-24-Methylene- d -Homocholesta-8,14-Dien-3β-ol in Ustilago maydis

“…In U. maydis the removal of the C-14 methyl is probably the first step in the reaction sequence from 24-methylenedihydrolanosterol to ergosterol (22). It has been postulated that the removal of the C-14 methyl group results in a C-14 (15) (16) and triforine (22) lead to a large accumulation of the C-4,C-14 methyl sterols (obtusifoliol and 24-methylenedihydrolanosterol) and a gradual buildup of 14a-methylergosta-8,24(28)-dienol. Our data indicate that once the C-14 methyl is removed, C-4 demethylation proceeds rapidly even though the resulting C-14(15) double bond is not reduced.…”

“…Component A25822B (15-aza-24-methylene-Dhomocholesta-8,14-dien-3,B-ol) (Fig. 1A) proved to be not only the major fraction of the complex (15) but also the most active antibiotic (10). Topical application of A25822B reduced the severity of Trichophyton mentagrophytes-induced lesions in guinea pigs.…”