NOESY and DFT-GIAO Calculations Reveal Pervasive Errors in C20 Configurations of Taraxastane-3,20-diols: Proposals to Improve NMR Structure Determinations.

The current scientific consensus generally assigns incorrect C20 configurations to taraxastane-3β,20-diols and their oxo derivatives reported in 32 articles. Definitive configurations established here by NOESY, quantum mechanical NMR predictions, and C NMR γ-gauche effects warrant updating of the many erroneous structures. Suggestions to improve NMR structure determinations of such nonpolar polycyclic natural products include acquiring 2D spectra at higher resolution, suppressing zero-quantum coherence in NOESY, and judging proposed NOE correlations against H-H distances.

Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination.

Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo.

Astertarone A Synthase from Chinese Cabbage Does Not Produce the C4-Epimer: Mechanistic Insights.

The Brassica rapa oxidosqualene cyclase Bra032185 makes 60% astertarone A (1) and 20 minor triterpenes (0.1-11%). Mechanistic analysis indicates the absence of an enol intermediate to generate the 4 S epimer of 1, unless workup involves saponification.

Torsional Failure of Carbon Fiber Composite Plates Versus Stainless Steel Plates for Comminuted Distal Fibula Fractures.

Background: Carbon fiber composite implants are gaining popularity in orthopedics, but with few independent studies of their failure characteristics under supra-physiologic loads. The objective of this cadaveric study was to compare torsional failure properties of bridge plating a comminuted distal fibula fracture with carbon fiber polyetheretherketone (PEEK) composite and stainless steel one-third tubular plates.

Methods: Comminuted fractures were simulated in 12 matched pairs of fresh-frozen human fibulas with 2-mm osteotomies located 3 cm proximal to the tibiotalar joint.

Are Isoursenol and γ-Amyrin Rare Triterpenes in Nature or Simply Overlooked by Usual Analytical Methods?

Among pentacyclic triterpenes commonly found in plants, γ-amyrin and isoursenol are seldom reported and considered rare in nature. It was hypothesized that these triterpenes are instead routinely overlooked due to inadequate spectral characterization. γ-Amyrin was prepared by HCOOH isomerization of α-amyrin, and isoursenol was isolated from products of a heterologously expressed oxidosqualene cyclase.

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.

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.

Trinorlupeol: a major nonsterol triterpenoid in Arabidopsis.

We report the structure determination of 20,29,30-trinorlup-18-en-3beta-ol (trinorlupeol) and establish this novel C 27 metabolite as a major nonsterol triterpenoid in Arabidopsis thaliana. Trinorlupeol was concentrated in cuticular waxes, notably in the plant stem, floral buds, and seedpods, but not in leaves. Based on expression data and functional characterization of A.

Arabidopsis camelliol C synthase evolved from enzymes that make pentacycles.

We establish by heterologous expression that the Arabidopsis thaliana oxidosqualene cyclase At1g78955 (CAMS1) makes camelliol C (98%), achilleol A (2%), and beta-amyrin (0.2%). CAMS1 is the first characterized cyclase that generates predominantly a monocyclic triterpene alcohol.

An oxidosqualene cyclase makes numerous products by diverse mechanisms: a challenge to prevailing concepts of triterpene biosynthesis.

The genome of the model plant Arabidopsis thaliana encodes 13 oxidosqualene cyclases, 9 of which have been characterized by heterologous expression in yeast. Here we describe another cyclase, baruol synthase (BARS1), which makes baruol (90%) and 22 minor products (0.02-3% each).

Stereochemistry of water addition in triterpene synthesis: the structure of arabidiol.

An oxidosqualene cyclase from Arabidopsis thaliana makes arabidiol, a tricyclic triterpene reported with indeterminate side-chain stereochemistry. We established the full structure of arabidiol through chemical degradation, NOE experiments, and molecular modeling. By examining the mechanistic constraints that govern water addition in triterpene synthesis, we further show how the stereochemistry of hydroxylation can generally be deduced a priori, why deprotonation is more common than hydroxylation, and why cyclases that perform hydroxylation also generate olefinic byproducts.

HEM dysplasia and ichthyosis are likely laminopathies and not due to 3beta-hydroxysterol Delta14-reductase deficiency.

Mutations of the lamin B receptor (LBR) have been shown to cause HEM dysplasia in humans and ichthyosis in mice. LBR is a bifunctional protein with both a lamin B binding and a sterol Delta(14)-reductase domain. It previously has been proposed that LBR is the primary sterol Delta(14)-reductase and that HEM dysplasia and ichthyosis are inborn errors of cholesterol synthesis.

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.

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.

A putative precursor of isomalabaricane triterpenoids from lanosterol synthase mutants.

[reaction: see text]. Known lanosterol synthase mutants produce monocyclic or tetracyclic byproducts from oxidosqualene. We describe Erg7 Tyr510 mutants that cause partial substrate misfolding and generate a tricyclic byproduct.

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity.

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood.

Enzymatic cyclization of dioxidosqualene to heterocyclic triterpenes.

Oxidosqualene cyclases normally produce triterpenes from 2,3-(S)-oxidosqualene (OS) but also can cyclize its minor companion (3S,22S)-2,3:22,23-dioxidosqualene (DOS). We explored DOS cyclization in plant triterpene synthesis using a recombinant lupeol synthase (LUP1) heterologously expressed in yeast. Incubation of LUP1 with 3S,22S-DOS gave epoxydammaranes epimeric at C20 and a 17,24-epoxybaccharane in a 4:2:3 ratio.

Structure and reactivity of the dammarenyl cation: configurational transmission in triterpene synthesis.

[reaction: see text] The dammarenyl cation (13) is the last common intermediate in the cyclization of oxidosqualene to a diverse array of secondary triterpene metabolites in plants. We studied the structure and reactivity of 13 to understand the factors governing the regio- and stereospecificity of triterpene synthesis. First, we demonstrated that 13 has a 17beta side chain in Arabidopsis thaliana lupeol synthase (LUP1) by incubating the substrate analogue (18E)-22,23-dihydro-20-oxaoxidosqualene (21) with LUP1 from a recombinant yeast strain devoid of other cyclases and showing that the sole product of 21 was 3beta-hydroxy-22,23,24,25,26,27-hexanor-17beta-dammaran-20-one.

Cholesterol import by Aspergillus fumigatus and its influence on antifungal potency of sterol biosynthesis inhibitors.

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.

Enzymatic synthesis of an indole diterpene by an oxidosqualene cyclase: mechanistic, biosynthetic, and phylogenetic implications.

Petromindole (1) is an unusual indole diterpene that bears a triterpene-like carbon skeleton, suggesting biogenesis from 3-(omega-oxido-geranylgeranyl)indole (4). We found that lupeol synthase (LUP1) from Arabidopsis thaliana cyclizes 4 to 1. Chiral HPLC comparisons of racemic 1 (from biomimetic cyclization of N-pivaloyl-4) with the LUP1 product and authentic 1 established the absolute stereochemistry of petromindole (3S) as that of cyclic triterpenes.

Programming of initial steps in bile acid synthesis by breast-feeding vs. formula-feeding in the baboon.

We tested the hypothesis that breast- vs. formula-feeding differentially affects the enzymatic activity of three sterol hydroxylases critical in the initial steps of bile acid formation. Thirty baboons were either breast-fed or formula-fed for the first 14 wk of life before weaning to baboon chow.

The implications of 7-dehydrosterol-7-reductase deficiency (Smith-Lemli-Opitz syndrome) to neurosteroid production.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation/mental retardation syndrome with an estimated incidence among individuals of European ancestry of 1 in 20000 to 1 in 30000. It is caused by inactivity of the enzyme 7-dehydrosterol-delta(7)-reductase, which catalyses the terminal transformation in cholesterol synthesis. Patients show not only an increased level of 7-dehydrocholesterol in blood and tissues, but also increased 8-dehydrocholesterol because of the presence of an active delta(8)-delta(7) isomerase.

Chromatographic behavior of oxygenated derivatives of cholesterol.

Oxygenated derivatives of cholesterol have important functions in many biochemical processes. These oxysterols are difficult to study because of their low physiological concentrations, the facile formation of cholesterol autoxidation artifacts, and lack of information on their chromatographic behavior. Focusing on metabolites and autoxidation products of cholesterol, we have documented the chromatographic mobilities of 35 oxysterols under a variety of conditions: eight solvent systems for thin-layer chromatography on silica gel, several mobile phases for reversed-phase high-performance liquid chromatography (HPLC), and two types of stationary phase for capillary gas chromatography (GC) using trimethylsilyl derivatives.

Identification of 7(8) and 8(9) unsaturated adrenal steroid metabolites produced by patients with 7-dehydrosterol-delta7-reductase deficiency (Smith-Lemli-Opitz syndrome).

Patients with Smith-Lemli-Opitz syndrome have impaired ability to synthesize cholesterol due to attenuated activity of 7-dehydrosterol-delta(7)-reductase which catalyses the final step in cholesterol synthesis. Accumulation of 7- and 8-dehydrocholesterol is a result of the disorder and potentially these sterols could be used as precursors of a novel class of delta(7) and delta(8) unsaturated adrenal steroids and their metabolites. In this study, we have analyzed urine from SLOS patients in the anticipation of characterizing such metabolites.