Recombinant squalene synthase. A mechanism for the rearrangement of presqualene diphosphate to squalene

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent reduction of PSPP to squalene (SQ). These reactions are the first committed steps in cholesterol biosynthesis. When recombinant SQase was incubated with FPP in the presence of dihydroNADPH (NADPH3, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed: dehydrosqualene (DSQ), a C30 analogue of phytoene; 10(S)-hydroxysqualene (HSQ), a hydroxy analogue of squalene; and rillingol (ROH), a cyclopropylcarbinyl alcohol formed by addition of water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement of PSPP to SQ (Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77). The structure and absolute stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two independent routes. Isolation of ROH from the enzyme-catalyzed reaction provides strong evidence for a cyclopropylcarbinyl-cyclopropylcarbinyl rearrangement in the biosynthesis of squalene. By comparing the SQase-catalyzed solvolysis of PSPP in the absence of NADPH3 to the reaction in the presence of NADPH3, it is apparent that the binding of the cofactor analogue substantially enhances the ability of SQase to control the regio- and stereochemistry of the rearrangements of PSPP.     

Synthesis and biological evaluation of quinuclidine derivatives incorporating phenothiazine moieties as squalene synthase inhibitors

Squalene synthase inhibitors have the potential to be superior hypocholesterolemic agents. A series of quinuclidine derivatives incorporating phenothiazine systems was synthesized in order to investigate the effects of their structure on the inhibition of hamster liver microsomal enzyme. (+/-)-3-(10-Methyl-10H-phenothiazin-3-ylmethoxy)quinuclidine hydrochloride (19) was the most potent inhibitor in this series with an IC(50) value of 0.12 microM. Oral dosing of compound 19 to hamsters demonstrated effective reduction of both plasma total cholesterol levels and plasma triglyceride levels. Compound 19 showed a reduced tendency to elevate plasma transaminase levels, an indicator of hepatotoxicity. Enantiomerically pure (-)-19, YM-53546, was found to be more potent than the corresponding (+)-enantiomer.     

Total synthesis of the squalene synthase inhibitor zaragozic acid C

Zaragozic acids and squalestatins were documented by Merck, Glaxo, and Tokyo Noko University/Mitsubishi Kasei Corporation as part of a program aimed at identifying novel inhibitors of squalene synthase, as well as farnesyl transferase. These natural products have attracted considerable attention from numerous synthetic chemists because of their therapeutic potential and novel architecture. This review highlights our total syntheses of zaragozic acid C by two convergent strategies. The key steps in our first-generation synthesis involve 1) simultaneous creation of the C4 and C5 quaternary stereocenters through the Sn(OTf)2-promoted aldol coupling reaction between the alpha-keto ester and silyl ketene thioacetal derived from L- and D-tartaric acids, respectively; and 2) construction of the bicyclic core structure via acid-catalyzed internal ketalization under kinetically controlled conditions. The second-generation strategy relies on a tandem carbonyl ylide formation/1,3-dipolar cycloaddition approach and features elongation of the C1 alkyl side chain through an olefin cross-metathesis as well as high convergency and flexibility.     

Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase

[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.     

Synthesis of E-C5-homolog of squalene

A stereospecific synthesis has been carried out for a monoisoprenolog of squalene by a cross-combination according to the Wurtz reaction of prenylmagnesium chloride with 3-hydroxyisosqualene tosylate.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 10, pp. 2293–2295, October, 1989.     

Synthesis of aryl-containing isoprenoids from 1,5-dimethylcycloocta-1,5-diene

A procedure has been developed for the synthesis of Pro-Drone homo analogs from the ozonolysis products of 1,5-dimethylcycloocta-1,5-diene and 1,5-dimethylcyclooctene.     

Evaluation of potential inhibitors of squalene synthase based on virtual screening and in vitro studies

Squalene synthase (SQS) is a potential target for hyperlipidemia treatment. To identify novel chemical scaffolds of SQS inhibitors, we generated 3D-QSAR pharmacophore models using HypoGen. The best quantitative pharmacophore model, Hypo 1, was selected for virtual screening using two chemical databases, Specs and Traditional Chinese Medicine database (TCM). The best-mapped hit compounds were then subjected to filtering by Lipinski^’s rule of five and docking studies to refine the hits. Finally, five compounds were selected from the top-ranked hit compounds for SQS inhibitory assay in vitro. Three of these compounds could inhibit SQS in vitro, and should be further evaluated pre-clinically as a treatment for hyperlipidemia.     

Synthesis of Z- and E-polyunsaturated isoprenoids with functional groups initiating electrophilic cyclization

Summary Separate olefination has been carried out for the carbonyl groups of 1,8-diketo-4-methyl-4Z-nonene and 1,12-diketo-4,8-dimethyl-4E, 8E-tridecadiene into 1,5-polyenes which were prepared for electrophilic cyclization.Deceased.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 4, pp. 821–823, April, 1981.     

Total synthesis and determination of the absolute stereochemistry of the squalene synthase inhibitors CJ-13,981 and CJ-13,982

The absolute and relative stereochemistries of the potent squalene synthase inhibitors CJ-13,981 and CJ-13,982 were determined to be 3S,4S by total synthesis of their antipodes using, as a key step, the diastereoselective alkylation of a chiral dioxolanone.     

Synthesis of 2-C-methyl-D-erythritol 4-phosphate: the first pathway-specific intermediate in the methylerythritol phosphate route to isoprenoids

[reaction: see text] 2-C-methyl-D-erythritol 4-phosphate (4), formed from 1-deoxy-D-xylulose 5-phosphate (3), is the first pathway-specific intermediate in the methylerythritol phosphate route for the biosynthesis of isoprenoid compounds in bacteria, algae, and plant chloroplasts. In this report, 4 was synthesized from 1,2-propanediol (7) in seven steps with an overall yield of 32% and in an enantiomeric excess of 78%.     

Synthesis of squalene epoxide and lanosterol analogues for a biosynthetic experiment

The syntheses of (21-¹⁴C)-2,3(RS)-oxide-2,6,10,15,19-pentamethyl-heneicosa-6E-10E, 14E,18Z-tetraene from squalene and of pure 20R and 20S 24,25,26,27-tetranorlanosteryl acetates from lanosteryl acetate are disclosed.     

Syntheses of (4,1-benzoxazepine-3-ylidene)acetic acid derivatives and their inhibition of squalene synthase

The (3,5-trans)-7-chloro-5-(2-chlorophenyl)-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid derivatives 1 have been previously identified as potent squalene synthase inhibitors. A series of (4,1-benzoxazepin-3-ylidene)acetic acid derivatives were synthesized and evaluated for their inhibition of rat and human squalene synthase, and the (E)-isomers were found to exhibit potent inhibitory activity, with the same potency as 4,1-benzoxazepine-3-acetic acid derivatives. In contrast the (Z)-isomers did not exhibit significant inhibitory activity, and the active conformation of the 4,1-benzoxazepine-3-acetic acid derivatives was deduced from the folded conformation of the (E)-isomers.     

Microbial enantioselective ester hydrolysis for the preparation of optically active 4,1-benzoxazepine-3-acetic acid derivatives as squalene synthase inhibitors

Microbial enantioselective ester hydrolysis for the preparation of optically active (3R,5S)-(-)-5-phenyl-4,1-benzoxazepine-3-acetic acid derivatives as potent squalene synthase inhibitors was investigated. Pseudomonas diminuta and Pseudomonas taetrolens hydrolyzed the racemic ethyl ester of the 5-(2-chlorophenyl) analogue to yield the (-)-carboxylic acid with excellent enantiomeric excess (>99% ee). We found that the (-)-enantiomer was an active inhibitor. Bulkiness of the ester moiety did not affect the enantioselectivity but did affect reactivity. The racemic ethyl ester of the 5-(2-methoxyphenyl) analogue, 5-(2,3-dimethoxyphenyl) analogue and 5-(2,4-dimethoxyphenyl) analogue were also hydrolyzed with Pseudomonas taetrolens to afford enantiomerically pure (-)-carboxylic acids in large scale. As another route to (3R,5S)-(-)-7-chloro-5-(2,3-dimethoxyphenyl)-1-neopentyl-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepine-3-acetic acid [(-)-1c], the earlier intermediate (-)-2-amino-5-chloro-alpha-(2,3-dimethoxyphenyl)benzyl alcohol [(-)-12] was successfully obtained by asymmetric hydrolysis of (+/-)-5-chloro-alpha-(2,3-dimethoxyphenyl)-2-pivaloylaminobenzyl acetate with Pseudomonas sp. S-13 with >99% ee in kilogram scale followed by alkaline treatment. The product (-)-12 was converted to (-)-1c without racemization.