2,3-Iminosqualene, a potent inhibitor of the enzymic cyclization of 2,3-oxidosqualene to sterols

Corey, E. J.; Montellano, Paul R. Ortiz de; Lin, Kang; Dean, P.D.G.

doi:10.1021/ja00987a089

Cited by 75 publications

(17 citation statements)

References 3 publications

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“…Inhibition constants were determined with Dixon plots . The (3 R , S )‐racemic mixture of iminosqualene ( 5 ) was chemically synthesized according to the reported method . The synthetic method for 5 is described in Fig.…”

Section: Resultsmentioning

confidence: 99%

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

2013

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b-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant b-amyrin synthase. The b-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni 2+ -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30°C, respectively. The kinetic parameters, K m and k cat , were determined to be 33.8 AE 0.53 lM and 46.4 AE 0.68 min À1 , respectively. To the best of our knowledge, there are no reports describing both K m and k cat for OSCs except for two examples of rat and bovine lanosterol synthases. The b-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k cat /K m ) (~10 3 -fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K i and IC 50 and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE (485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.Abbreviations EtAS, Euphorbia tirucalli b-amyrin synthase; OSC, oxidosqualene cyclase; SHC, squalene-hopene cyclase; THF, tetrahydrofuran; b-OG, b-octylglucoside.

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Section: Resultsmentioning

confidence: 99%

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

2013

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“…The obvious structural similarities have stimulated the synthesis and evaluation of many isoprenoid aziridines as inhibitors of terpene biosynthesis. For example, 2,3-epiiminosqualene, the aziridine analogue of 2,3-oxidosqualene, is an effective inhibitor of oxidosqualene cyclases 4. Sterol analogues bearing an aziridine ring in the side chain at the 24, 25 position are potent inhibitors of cholesterol biosynthesis in rat hepatoma cells,5a and they block sterol methyl transferase activity essential for plant development5b and fungal cell growth 5c,5d.…”

Section: Introductionmentioning

confidence: 99%

Scope and Mechanism of Intramolecular Aziridination of Cyclopent-3-enyl-methylamines to 1-Azatricyclo[2.2.1.0^2,6]heptanes with Lead Tetraacetate

Faraldos

Coates

2009

J. Am. Chem. Soc.

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A series of seven cyclopent-3-en-1-ylmethylamines bearing one, two, or three methyl substituents at the C2, C3, C4, or C α positions, including the unsubstituted parent, was accessed by ring-closing metatheses of α,α-diallylacetonitrile (or methallyl variants) and α,α-diallylacetone followed by hydride reductions, or by Curtius degradations of α,α-dimethyl-and 2,2,3-trimethylcyclopent-3-enylacetic acids. Oxidation of the primary amines with Pb(OAc) 4 in CH 2 Cl 2 , CHCl 3 or benzene in the presence of K 2 CO 3 effected efficient intramolecular aziridinations, in all cases except the α-methyl analogue (16), to form the corresponding 1-azatricyclo[2.2.1.0 2,6 ]heptanes, including the novel monoterpene analogues, 1-azatricyclene and the 2-azatricyclene enantiomers. The cumulative rate increases of aziridination reactions observed by 1 H NMR spectroscopy in CDCl 3 resulting from the presence of one or two methyl groups on the cyclopentene double bond, in comparison to the rate of the unsubstituted parent amine (1 : 17.5 : >280), indicate a highly electrophilic intermediate as the nitrene donor and a symmetrical aziridine-like transition state. A mechanism is outlined in which the amine displaces an acetate ligand from Pb(OAc) 4 to form a lead(IV) amide intermediate RNHPb(OAc) 3 proposed as the actual aziridinating species.

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“…NH‐Aziridine derivatives of isoprenoid polyenes are potential candidates for the selective inhibition of the biosynthesis of sterol …”

Section: Nh‐aziridination By Using An N‐acetoxyamine Quinazolone Reagentmentioning

confidence: 99%

Unprotected Aziridines: A Synthetic Overview

2017

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Unprotected (NÀH) aziridines are the key components in several biologically active natural products and important buildingb locks in organic synthesis. However,t he synthesis of unprotecteda ziridines is much more difficult than their oxygen counterparts (epoxides). Therefore, the development of efficient routes for their synthesis represents an attractive task fororganic chemists and, as aresult, avariety of methods have been developed. In this Focus Review, we have described the various approaches that have been reportedf or the synthesis of NH-aziridines.

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2,3-Iminosqualene, a potent inhibitor of the enzymic cyclization of 2,3-oxidosqualene to sterols

Cited by 75 publications

References 3 publications

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

Scope and Mechanism of Intramolecular Aziridination of Cyclopent-3-enyl-methylamines to 1-Azatricyclo[2.2.1.0^2,6]heptanes with Lead Tetraacetate

Unprotected Aziridines: A Synthetic Overview

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2,3-Iminosqualene, a potent inhibitor of the enzymic cyclization of 2,3-oxidosqualene to sterols

Cited by 75 publications

References 3 publications

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

Scope and Mechanism of Intramolecular Aziridination of Cyclopent-3-enyl-methylamines to 1-Azatricyclo[2.2.1.02,6]heptanes with Lead Tetraacetate

Unprotected Aziridines: A Synthetic Overview

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Scope and Mechanism of Intramolecular Aziridination of Cyclopent-3-enyl-methylamines to 1-Azatricyclo[2.2.1.0^2,6]heptanes with Lead Tetraacetate