Harzianone Biosynthesis by the Biocontrol Fungus Trichoderma.

Analysis of the volatile terpenes produced by seven fungal strains of the genus Trichoderma by use of a closed-loop stripping apparatus (CLSA) revealed a common production of harzianone, a bioactive, structurally unique diterpenoid consisting of a fused tetracyclic 4,7,5,6-membered ring system. The terpene cyclization mechanism was studied by feeding experiments using selectively 13 C- and 2 H-labeled synthetic mevalonolactone isotopologues, followed by analysis of the incorporation patterns by 13 C NMR spectroscopy and GC/MS. The structure of harzianone was further supported from a 13 C,13 C COSY experiment of the in-vivo-generated fully 13 C-labeled diterpene.

Synthesis of mevalonate- and fluorinated mevalonate prodrugs and their in vitro human plasma stability.

The mevalonate pathway is essential for the production of many important molecules in lipid biosynthesis. Inhibition of this pathway is the mechanism of statin cholesterol-lowering drugs, as well as the target of drugs to treat osteoporosis, to combat parasites, and to inhibit tumor cell growth. Unlike the human mevalonate pathway, the bacterial pathway appears to be regulated by diphosphomevalonate (DPM). Enzymes in the mevalonate pathway act to produce isopentenyl diphosphate, the product of the DPM decarboxylase reaction, utilize phosphorylated (charged) intermediates, which are poorly bioavailable. It has been shown that fluorinated DPMs (6-fluoro- and 6,6,6-trifluoro-5-diphosphomevalonate) are excellent inhibitors of the bacterial pathway; however, highly charged DPM and analogs are not bioavailable. To increase cellular permeability of mevalonate analogs, we have synthesized various prodrugs of mevalonate and 6-fluoro- and 6,6,6-trifluoromevalonate that can be enzymatically transformed to the corresponding DPM or fluorinated DPM analogs by esterases or amidases. To probe the required stabilities as potentially bioavailable prodrugs, we measured the half-lives of esters, amides, carbonates, acetals, and ketal promoieties of mevalonate and the fluorinated mevalonate analogs in human blood plasma. Stability studies showed that the prodrugs are converted to the mevalonates in human plasma with a wide range of half-lives. These studies provide stability data for a variety of prodrug options having varying stabilities and should be very useful in the design of appropriate prodrugs of mevalonate and fluorinated mevalonates.

Synthesis and anti-hypercholesterolemic evaluations of calcium salts of 4-substituted quinoline-based mevalonic acid.

A series of calcium (3R,5S,6E)-7-[4,6,7,8-substituted-quinoline-3-yl]-3,5-dihydroxy-hept-6-enoates was synthesized from the lactones, and the anti-hypercholesterolemic evaluation in quails was presented in this report. It was found that most of the compounds significantly decreased levels of total cholesterol and low-density lipoprotein. 2e showed better hypolipidemic effect than atorvastatin, and 2d and 2j exhibited comparable efficacy to atorvastatin. These three compounds were selected as the hypocholesterolemic candidates for further evaluation.

Synthesis and HMG-CoA reductase inhibition of 2-cyclopropyl-4-thiophenyl-quinoline mevalonolactones.

A novel series of 2-cyclopropyl-4-thiophenyl quinoline-based mevalonolactones were synthesized from the substituted anilines by several reactions. Among them, (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(4-fluoro-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1d), (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4-(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1f) and (4R,6S)-6-[(E)-2-(2-cyclopropyl-6-fluoro-4,7-di(3-methoxy-thiophenyl)-quinoline-3-yl)-ethenyl]-3,4,5,6-tetrahydro-4-hydroxy-2H-pyran-2-one (1q) showed potent HMG-CoA reductase inhibitory activity comparable with pitavastatin.

Bifunctional inhibitors of mevalonate kinase and mevalonate 5-diphosphate decarboxylase.

[structure: see text] A bifunctional inhibitor of mevalonate kinase and mevalonate 5-diphosphate decarboxylase was synthesized. Both enzymes are in the cholesterol biosynthetic pathway and play an important role in regulating cholesterol biosynthesis. The molecule may become a useful lead compound for further development for treating cardiovascular disease and cancer. This study provides a novel example of a single inhibitor blocking two sequential steps simultaneously in the cholesterol biosynthetic pathway.

Multienzyme mevalonate pathway bioreactor.

The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.

Synthetic applications of epoxide hydrolases.

There have been several recent advances in the area of biocatalysed hydrolytic kinetic resolution of epoxides using 'newly discovered' enzymes (i.e. epoxide hydrolases). These biocatalysts, two of which will become commercially available in the near future, appear to be highly promising tools for fine organic synthesis, as they enable the preparation of various epoxides and/or their corresponding diols in enantiopure form.

Determination of mevalonolactone in capsules by capillary gas-liquid chromatography.

A wide bore capillary gas chromatographic method was developed to determine mevalonolactone in capsule formulations. The method uses beta,beta-dimethyl-gamma-hydroxymethyl-gamma- butyrolactone as an internal standard and has been validated for its accuracy, precision and linearity. The method has been applied for stability testing of the capsule formulation. High-performance liquid chromatographic and gas chromatographic studies demonstrated cyclization of mevalonic acid (open-chain form) to mevalonolactone (cyclic form) under the described gas chromatography conditions. Mass spectrometric analysis indicated that mevalonolactone prepared in water or an organic solvent emerged from the gas chromatographic column as the intact cyclic lactone.

Inhibitors of cholesterol biosynthesis. 2. 1,3,5-trisubstituted [2-(tetrahydro-4-hydroxy-2-oxopyran-6-yl)ethyl]pyrazoles.

A series of 1,3,5-trisubstituted pyrazole mevalonolactones were prepared and evaluated for their ability to inhibit the enzyme HMG-CoA reductase in vitro. Since previous studies suggested that the 5-(4-fluorophenyl) and 3-(1-methylethyl) substituents afforded optimum potency, attention was focused on variations in position 1 of the pyrazole ring. Biological evaluation of analogues bearing a variety of 1-substituents suggested that, although most substituents were tolerated, none afforded an advantage over phenyl, which exhibited potency comparable to that of compactin in vitro.

Enzymatic synthesis of mevalonate-5-(2-thiodiphosphate).

Mevalonate-5-(2-thiodiphosphate), a substrate analog for diphosphomevalonate decarboxylase, has been enzymatically prepared from mevalonate-5-phosphate and adenosine-5'-0-(3-thiotriphosphate) using phosphomevalonate kinase as a catalyst, in a 37% yield. The substrate properties of the synthesized compound are compared to those of the normal substrate of the enzyme.

Improved procedures for the synthesis of phosphomevalonate and for the assay and purification of pig liver phosphomevalonate kinase.

An improved procedure for the synthesis of phosphomevalonate using excess free ATP4-, and phenyl agarose to remove contaminating nucleotides, is described. A high-voltage electrophoresis assay, which separates phosphomevalonate from mevalonate 5-diphosphate at pH 3.5, was developed for the assay of phosphomevalonate kinase (ATP:5-phosphomevalonate phosphotransferase, EC 2.7.4.2). High-voltage electrophoresis, at pH 5, could also be used for the separation of mevalonate 5-diphosphate from isopentenyl diphosphate. An alternative method for the purification of phosphomevalonate kinase from pig liver was also developed. The high-voltage electrophoresis assay was used to reassess the metal ion and nucleotide specificity of the pig liver phosphomevalonate kinase. ATP could be partially replaced by ITP and GTP and poorly by CTP and UTP. Apparent activation of the enzyme by free ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224).

Synthesis of 3-hydroxy-3-cyclohexylbutyric acid derivatives. 2. Cyclic analogues of mevalonic acid.

The sodium salt of (Z)-3-hydroxy-3-(2-hydroxycyclohexyl)butyric acid (I) and its lactone (II) were prepared through the corresponding tert-butyl ester by hydrogenation, over Rh/Al2O3 catalyst, of the phenyl ring of tert-butyl 3-hydroxy-3-(2-hydroxyphenyl)butyrate (III). (Z)-3-Hydroxy-3-(2-methoxycyclohexyl)butyric acid was prepared similarly. (Z)-4-Methyloctahydro-2H-1-benzopyran-2-one was prepared by hydrogenation, over Rh/Al2O3 catalyst, of 4-methylcoumarine, prepared in turn from III by a one-pot procedure comprising hydrolysis, lactonization, and dehydration. The above compounds inhibit acetate incorporation in cholesterol and fatty acids in rat liver slices at 5 X 10(-3) M, but they lack specific inhibitory activity on HMG-CoA reductase.