Homobotcinolide: a biologically active natural homolog of botcinolide from Botrytis cinerea.

A novel natural product exhibiting biological activity was isolated from a strain of Botrytis cinerea that had infected raspberry fruit (Rubus ideaus). Liquid fermentation and bioassay-directed fractionation of the organism yielded a compound with molecular formula C22H38O8 that is trivially named homobotcinolide. It significantly inhibited etiolated wheat coleoptile growth. Greenhouse-grown bean, corn, and tobacco plants were also affected by exogenous application of homobotcinolide, severe chlorosis and necrosis being exhibited in corn. The compound is a polyhydroxylated nonalactone esterified with 4-hydroxy-2-decenoic acid.

The structural requirements of sterols for membrane function in Saccharomyces cerevisiae.

Cultures of Saccharomyces cerevisiae strain GL7 auxotrophic for sterol were incubated with a series of sterols and sterol-like molecules (tetracyclic and pentacyclic triterpenoids) in order to determine the structural requirements of sterols for bulk membrane function. For growth support, the 3 beta-OH group could not be replaced by H, OMe, OBu, NH2, NHOH, OAc, keto, or 3 alpha-OH. A methyl group at C-14 was neither deleterious nor essential for activity. Removal of the C-4 geminal methyl group was obligatory for activity. Thus, no sterol-like triterpenoid supported growth (e.g., tetrahymanol, lanosterol, and cycloartenol). Growth support required a sterol with the longest methylene segment extending from C-20 not to exceed six contiguous C-atoms and the stereochemistry must be C-20 R. No significance could be attributed to branching at C-20 (i.e., to C-21), C-24 (when alkylated), or C-25 (regarding the isopropyl group). Double bonds in the nucleus were not essential for activity since cholestanol supported growth. In several incubations, the addition of trace levels of dietary ergosterol (0.5 microgram/ml) to the medium was necessary to promote growth and transformation of the bulk sterol to a membrane competent sterol(s).

Regulation of sterol biosynthesis in sunflower by 24(R,S),25-epiminolanosterol, a novel C-24 methyl transferase inhibitor.

Whereas sitosterol and 24(28)-methylene cycloartanol were competitive inhibitors (with Ki = 26 microM and 14 microM, respectively), 24(R,S)-25-epiminolanosterol was found to be a potent non-competitive inhibitor (Ki = 3.0 nM) of the S-adenosyl-L-methionine-C-24 methyl transferase from sunflower embryos. Because the ground state analog, 24(R,S)-oxidolanosterol, failed to inhibit the catalysis and 25-azalanosterol inhibited the catalysis with a Ki of 30 nM we conclude that the aziridine functions in a manner similar to the azasteriod (Rahier, A., et al., J. Biol. Chem. (1984) 259, 15215) as a transition state analog mimicking the carbonium intermediate found in the normal transmethylation reaction. Additionally, we observed that the aziridine inhibited cycloartenol metabolism (the preferred substrate for transmethylation) in cultured sunflower cells and cell growth.

Sterol phylogenesis and algal evolution.

The stereochemistry of several sterol precursors and end products synthesized by two fungal-like micro-organisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and 1H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecycloartanol, ergosterol, protothecasterol, 4alpha-methylergostanol, 4alpha-methylclionastanol, clionastanol, 24beta-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I [2-3H]lanosterol, [2-3H]cycloartenol, [24-3H]lanosterol, and [methyl-2H3]methionine and by feeding to II [methyl-2H3]methionine. The results demonstrate that the 24beta configuration is formed by different alkylation routes in I and II. The Delta25(27) route operates in I while the Delta24(28) route operates in II. Based on what is known in the literature regarding sterol distribution and phylogenesis together with our findings that the stereochemical outcome of squalene oxide cyclization leads to the production of cycloartenol rather than lanosterol (characteristic of the fungal genealogy) and the chirality of the C-24 alkyl group is similar in the two nonphotosynthetic microbes (beta oriented), we conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors.

Comparison of the chromatographic properties of sterols, select additional steroids and triterpenoids: gravity-flow column liquid chromatography, thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography.

The chromatographic properties of approximately 100 sterols, select steroids of plant origin (sapogenins and steroidal alkaloids) and triterpenoids has been evaluated in this laboratory by monitoring their elution characteristics in adsorption (gravity column and thin-layer methods with and without the addition of silver nitrates), gas and reversed-phase high-performance liquid chromatography. The utility of each methodology to act in one or another chromatographic mode-separation, radio-chemical purification, quantitation and structural elucidation, is discussed. The importance of the tilt of the -OH group at C-3 as well as the polarity, size, an shape of the rest of the molecule as it effects the hydrogen-bonding ability of the -OH group is demonstrated through changes in chromatographic behavior that result from the step-wise introduction of double bonds, methyl, bromo, oxygen, nitrogen and cyclopropyl groups into 5 alpha-cholestanol. An independent aid in the structure identification and quantitation of the compounds was use of a multiple-wavelength diode array detector in which different wavelengths of the UV spectrum (200-400 nm) were simultaneously monitored following passage of the sample through a reversed-phase C18 column.