Quanbo Xiong

Mechanistic insights into triterpene synthesis from quantum mechanical calculations. Detection of systematic errors in B3LYP cyclization energies

Most quantum mechanical studies of triterpene synthesis have been done on small models. We calculated mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* energies for many C30H51O+ intermediates to establish the first comprehensive energy profiles for the cationic cyclization of oxidosqualene to lanosterol, lupeol, and hopen-3beta-ol. Differences among these 3 profiles were attributed to ring strain, steric effects, and proton affinity. Modest activation energy barriers and the ample exothermicity of most annulations indicated that the cationic intermediates rarely need enzymatic stabilization. The course of reaction is guided by hyperconjugation of the carbocationic 2p orbital with parallel C-C and C-H bonds. Hyperconjugation for cations with a horizontal 2p orbital (in the plane of the ABCD ring system) leads to annulation and ring expansion. If the 2p orbital becomes vertical, hyperconjugation fosters 1,2-methyl and hydride shifts. Transition states leading to rings D and E were bridged cyclopropane/carbonium ions, which allow ring expansion/annulation to bypass formation of undesirable anti-Markovnikov cations. Similar bridged species are also involved in many cation rearrangements. Our calculations revealed systematic errors in DFT cyclization energies. A spectacular example was the B3LYP/6-311+G(2d,p)//B3LYP/6-31G* prediction of endothermicity for the strongly exothermic cyclization of squalene to hopene. DFT cyclization energies for the 6-311+G(2d,p) basis set ranged from reasonable accuracy (mPW1PW91, TPSSh with 25% HF exchange) to underestimation (B3LYP, HCTH, TPSS, O3LYP) or overestimation (MP2, MPW1K, PBE1PBE). Despite minor inaccuracies, B3LYP/6-31G* geometries usually gave credible mPW1PW91 single-point energies. Nevertheless, DFT energies should be used cautiously until broadly reliable methods are established.

Cholesterol Import by Aspergillus fumigatus and Its Influence on Antifungal Potency of Sterol Biosynthesis Inhibitors

ABSTRACT High mortality rates from invasive aspergillosis in immunocompromised patients are prompting research toward improved antifungal therapy and better understanding of fungal physiology. Herein we show that Aspergillus fumigatus, the major pathogen in aspergillosis, imports exogenous cholesterol under aerobic conditions and thus compromises the antifungal potency of sterol biosynthesis inhibitors. Adding serum to RPMI medium led to enhanced growth of A. fumigatus and extensive import of cholesterol, most of which was stored as ester. Growth enhancement and sterol import also occurred when the medium was supplemented with purified cholesterol instead of serum. Cells cultured in RPMI medium with the sterol biosynthesis inhibitors itraconazole or voriconazole showed retarded growth, a dose-dependent decrease in ergosterol levels, and accumulation of aberrant sterol intermediates. Adding serum or cholesterol to the medium partially rescued the cells from the drug-induced growth inhibition. We conclude that cholesterol import attenuates the potency of sterol biosynthesis inhibitors, perhaps in part by providing a substitute for membrane ergosterol. Our findings establish significant differences in sterol homeostasis between filamentous fungi and yeast. These differences indicate the potential value of screening aspergillosis antifungal agents in serum or other cholesterol-containing medium. Our results also suggest an explanation for the antagonism between itraconazole and amphotericin B, the potential use of Aspergillus as a model for sterol trafficking, and new insights for antifungal drug development.

Protostadienol biosynthesis and metabolism in the pathogenic fungus Aspergillus fumigatus.

Details of the fungal biosynthetic pathway to helvolic acid and other fusidane antibiotics remain obscure. During product characterization of oxidosqualene cyclases in Aspergillus fumigatus, we found the long-sought cyclase that makes (17Z)-protosta-17(20),24-dien-3beta-ol, the precursor of helvolic acid. We then identified a gene cluster encoding the pathway to helvolic acid, which is controlled by a transcription regulator (LaeA) associated with fungal virulence. Evidence regarding the evolutionary origin and taxonomic distribution of fusidane biosynthesis is also presented.

The liebermann-burchard reaction : Sulfonation, desaturation, and rearrangment of cholesterol in acid

In the Liebermann–Burchard (LB) colorimetric assay, treatment of cholesterol with sulfuric acid, acetic anhydride, and acetic acid elicits a blue color. We studied the reactivity of cholesterol under LB conditions and provide definitive NMR characterization for approximately 20 products, whose structure and distribution suggest the following mechanistic picture. The major reaction pathways do not involve cholestadienes, i-steroids, or cholesterol dimers, as proposed previously. Instead, cholesterol and its acetate and sulfate derivatives undergo sulfonation at a variety of positions, often with skeletal rearrangements. Elimination of an SO3H group as H2SO3 generates a new double bond. Repetition of this desaturation process leads to polyenes and ultimately to aromatic steroids. Linearly conjugated polyene cations can appear blue but form too slowly to account for the LB color response, whose chemical origin remains unidentified. Nevertheless, the classical polyene cation model is not excluded for Salkowski conditions (sulfuric acid), which immediately generate considerable amounts of cholesta-3,5-diene. Some rearrangements of cholesterol in H2SO4 resemble the diagenesis pathways of sterols and may furnish useful lipid biomarkers for characterizing geological systems.

Product profile of PEN3: the last unexamined oxidosqualene cyclase in Arabidopsis thaliana.

The triterpene product profile is reported for At5g36150 (PEN3), the last unexamined oxidosqualene cyclase in the reference plant Arabidopsis thaliana. PEN3 makes tirucalla-7,24-dien-3beta-ol ( approximately 85%) and several minor products. Also discussed are the unexpectedly facile convergent evolution of another Arabidopsis tirucalladienol synthase (LUP5), mechanistic origins of the 20S configuration, and active-site remodeling necessary to accommodate the 17alpha side chain. This work marks the first completed functional characterization of all triterpene synthases in a higher plant.

A colorimetric assay for 7-dehydrocholesterol with potential application to screening for Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS; MIM 270400) is a genetic disorder characterized by hypocholesterolemia and elevated 7-dehydrocholesterol (7DHC) levels resulting from mutations affecting 7-dehydrocholesterol reductase. We describe a colorimetric assay for 7DHC with potential application to large-scale screening for SLOS. Reaction of 7DHC and its esters with the Liebermann-Burchard reagent resulted in a brief initial absorbance at 510 nm (pink color) followed by an absorbance at 620 nm (blue color) after 2 min, while cholesterol samples were essentially colorless. The assay could identify typical SLOS blood samples by their pink color and increased absorbance at 620 nm after 2 min. Colorimetric identification of mild SLOS cases requires monitoring of the transient absorbance at 510 nm, which must be detected immediately after rapid, consistent mixing of the reagents. The need for special mixing devices and rigorous validation precludes sporadic use of the assay for diagnosing suspected SLOS cases. We also studied the stability of 7DHC in dried SLOS blood spots on Guthrie cards, which are widely used for archiving neonatal blood. Decomposition of 7DHC was effectively retarded by storage at low temperature and by precoating of the cards with antioxidants. The combined results provide a foundation for development of a simple, automated test for SLOS screening.