Abstract:A theoretical conformational study based on density functional calculations provides evidence that the sterol and nonsterol cyclizations of squalene to triterpenes are controlled by conformational effects as has been previously suggested. It was found that different conformers of a model system of squalene give rise to the chair−boat conformation found in the steroids and the chair−chair conformation of the pentacyclic 3‐deoxytriterpenes for their A and B rings. It is suggested that the enzymes play a key role… Show more
“…The stabilizing effect of the 20-oxa substitution comes into play only as the annulation process nears completion and thus does not influence the activation energy. The transition states and activation energies closely resemble computational results for protosteryl cation formation by Hess,7d who first proposed bridged transition states in C-ring expansion/D-ring formation as an alternative to anti-Markovnikov intermediates. ,7b,j,p 2 Changes in energy (A) and geometry (B) from path calculations modeling 17β-dammarenyl cation formation with and without the 20-oxygen. Energies in panel A are from mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* calculations.…”
Section: Resultssupporting
confidence: 69%
“…We optimized likely transition-state structures leading to the hopyl and lupyl cations ( 5 and 15 ). Their geometries, shown in Figure , indicate that the transition state for D-ring expansion/E-ring formation, like that of C-ring expansion/D-ring formation,7d occurs during ring expansion and before closure of the new ring. These transition-state structures are cyclopropane/carbonium ions and represent lower energy pathways than the 6−6−6 and 6−6−6−6 secondary carbocations that have often been invoked as intermediates in (oxido)squalene cyclization 29a…”
Section: Resultsmentioning
confidence: 99%
“…The classic scheme for understanding triterpene biosynthesis 1a,4,5 accounts for known triterpene skeletons 3 through application of basic rules governing the reactivity of cations. Nevertheless, much greater complexity than originally envisioned has been revealed by recent studies on molecular modeling, protein crystallography, , biomimetic synthesis, mechanism, , natural product isolation, , evolutionary origins, mutant cyclases, inhibitors, substrate analogues, and characterization of cloned cyclases . Molecular modeling and experiment have cast doubt on the seminal hypothesis that cyclization is a concerted process .…”
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 17 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 3 -hydroxy- 22,23,24,25,26,27-hexanor-17 -dammaran-20-one. Quantum mechanical calculations were carried out on gas-phase models to show that the 20-oxa substitution has negligible effect on substrate binding and on the activation energies of reactions leading to either C17 epimer of 13. Further molecular modeling indicated that, because of limited rotational freedom in the cyclase active site cavity, the C17 configuration of the tetracyclic intermediate 13 can be deduced from the angular methyl configuration of the pentacyclic or 6-6-6-6 tetracyclic product. This rule of configurational transmission aided in elucidating the mechanistic pathway accessed by individual cyclases. Grouping of cyclases according to mechanistic and taxonomic criteria suggested that the transition between pathways involving 17R and 17 intermediates occurred rarely in evolutionary history. Two other mechanistic changes were also rare, whereas variations on cation rearrangements evolved readily. This perspective furnished insights into the phylogenetic relationships of triterpene synthases.
“…The stabilizing effect of the 20-oxa substitution comes into play only as the annulation process nears completion and thus does not influence the activation energy. The transition states and activation energies closely resemble computational results for protosteryl cation formation by Hess,7d who first proposed bridged transition states in C-ring expansion/D-ring formation as an alternative to anti-Markovnikov intermediates. ,7b,j,p 2 Changes in energy (A) and geometry (B) from path calculations modeling 17β-dammarenyl cation formation with and without the 20-oxygen. Energies in panel A are from mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* calculations.…”
Section: Resultssupporting
confidence: 69%
“…We optimized likely transition-state structures leading to the hopyl and lupyl cations ( 5 and 15 ). Their geometries, shown in Figure , indicate that the transition state for D-ring expansion/E-ring formation, like that of C-ring expansion/D-ring formation,7d occurs during ring expansion and before closure of the new ring. These transition-state structures are cyclopropane/carbonium ions and represent lower energy pathways than the 6−6−6 and 6−6−6−6 secondary carbocations that have often been invoked as intermediates in (oxido)squalene cyclization 29a…”
Section: Resultsmentioning
confidence: 99%
“…The classic scheme for understanding triterpene biosynthesis 1a,4,5 accounts for known triterpene skeletons 3 through application of basic rules governing the reactivity of cations. Nevertheless, much greater complexity than originally envisioned has been revealed by recent studies on molecular modeling, protein crystallography, , biomimetic synthesis, mechanism, , natural product isolation, , evolutionary origins, mutant cyclases, inhibitors, substrate analogues, and characterization of cloned cyclases . Molecular modeling and experiment have cast doubt on the seminal hypothesis that cyclization is a concerted process .…”
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 17 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 3 -hydroxy- 22,23,24,25,26,27-hexanor-17 -dammaran-20-one. Quantum mechanical calculations were carried out on gas-phase models to show that the 20-oxa substitution has negligible effect on substrate binding and on the activation energies of reactions leading to either C17 epimer of 13. Further molecular modeling indicated that, because of limited rotational freedom in the cyclase active site cavity, the C17 configuration of the tetracyclic intermediate 13 can be deduced from the angular methyl configuration of the pentacyclic or 6-6-6-6 tetracyclic product. This rule of configurational transmission aided in elucidating the mechanistic pathway accessed by individual cyclases. Grouping of cyclases according to mechanistic and taxonomic criteria suggested that the transition between pathways involving 17R and 17 intermediates occurred rarely in evolutionary history. Two other mechanistic changes were also rare, whereas variations on cation rearrangements evolved readily. This perspective furnished insights into the phylogenetic relationships of triterpene synthases.
“…To date, no computational study on the overall mechanism of OSC has been reported, except that the first step (conversion R → 1 ) was studied by QM calculations on small models . The mechanism of squalene-hopene cyclase (SHC), a closely related enzyme, has been investigated in a number of computational studies, − where the ring formation steps have been discussed in great detail. In the current work, we are interested in the product specificity at the hydride/methyl-shifting stage, which was never previously studied.…”
Oxidosqualene-lanosterol cyclase (OSC) is a key enzyme in the biosynthesis of cholesterol. The catalytic mechanism and the product specificity of OSC have herein been studied using QM/MM calculations. According to our calculations, the protonation of the epoxide ring of oxidosqualene is rate-limiting. Wild-type OSC (which generates lanosterol), and the mutants H232S (which generates parkeol) and H232T (which generates protosta-12,24-dien-3-β-ol) were modeled, in order to explain the product specificity thereof. We show that the product specificity of OSC at the hydride/methyl-shifting stage is unlikely to be achieved by the stabilization of the cationic intermediates, as the precursor of lanosterol is in fact not the most stable cationic intermediate for wild-type OSC. The energy barriers for the product-determining conversions are instead found to be related to the product specificity of different OSC mutants, and we thus suggest that the product specificity of OSC is likely to be controlled by kinetics, rather than thermodynamics.
“…For this cyclization to be concerted (6‐6‐5, chair–boat formation), a conformation of the squalene oxide must exist that once protonated has the electrophilic CC double bonds positioned to interact with the developing positively charged carbon atoms. Our earlier studies on the individual cyclizations have shown that for a concerted cyclizations to occur, the conformation about the key CC bond must not exist as a minimum if a positive charge is developed in the vicinity of the achimerically assisting double bond that supplies the electrons for the newly forming CC σ bond 9c,d…”
Konzertiertes ABC: Für die Bildung der Ringe A–C in der biosynthetischen Umwandlung von Squalenoxid in das Prosterol‐Kation wurde ein konzertierter, wenn auch stark asynchroner Pfad lokalisiert, durch den der Ring B in der erforderlichen Bootkonformation erhalten wird.
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