Synthesis of 2-(2-Hydroxyethoxy)-3-hydroxysqualene and Characterization of Its Anti-Inflammatory Effects.

Squalene (SQ), a natural precursor of many steroids, can inhibit tumor progression and decrease serum cholesterol levels. However, it is difficult to discern the effect of highly active molecules in the treatment of diseases because not enough active compounds reach the site of pathology in crowded biosystems. Therefore, it is necessary to design artificial probes that work effectively within crowded systems. In this study, to facilitate cell penetration, the ethylene glycol moiety (used as a probe) was chemically added to SQ to form 2-(2-hydroxyethoxy)-3-hydroxysqualene (HEHSQ). HEHSQ was prepared from 2,3-epoxysqualene and characterized by Rf, FT-IR, 1H NMR, 13C NMR, and high-resolution mass spectrometry. We then evaluated the anti-inflammatory effects of SQ and HEHSQ on lipopolysaccharide- (LPS-) stimulated RAW264.7 murine macrophages. To determine the effect of SQ and HEHSQ on the viability of RAW264.7 cells, an MTT assay was performed. To quantify the anti-inflammatory effect of SQ and HEHSQ, we measured nitric oxide (NO) production, gene expression, and secretion of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and chemokine C-C motif chemokine 2 (CCL2) in LPS-stimulated RAW264.7 cells using an in vitro inflammatory model. 2,3-Epoxysqualene was prepared according to a reported methodology. The reaction of 2,3-epoxysqualene and ethylene glycol in 2-propanol produced 49% HEHSQ. MTT results showed that 10 and 100 µg/mL HEHSQ treatment decreased cell viability, whereas SQ treatment (1-100 µg/mL) did not have any effect on viability. SQ (100 µg/mL) and HEHSQ (1 µg/mL) treatment significantly reduced the production of LPS-stimulated NO and decreased the expression and secretion of proinflammatory TNF-α and CCL2. Therefore, our results suggested that the anti-inflammatory effects of HEHSQ are 100 times higher than that of unmodified SQ. To the best of our knowledge, this study has demonstrated for the first time that HEHSQ can be potentially used as a safe alternative treatment to anti-inflammatory drugs.

Two new convenient syntheses of 14C-squalene from turbinaric acid.

Carbon-14 labeled (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, also known as squalene, was synthesized as a tool for pharmacokinetic studies. Two simple and efficient labeling approaches were developed to give [2-14 C]-squalene and [3-14 C]-squalene from a halogenated precursor derived from turbinaric acid. They were obtained in 13.5% radiochemical yield in 6 steps and in 38% radiochemical yield in 3 steps respectively from carbon-14 labeled potassium cyanide with a radiochemical purity higher than 98% in both cases.

Biomimetic Platinum-Promoted Polyene Polycyclizations: Influence of Alkene Substitution and Pre-cyclization Conformations.

Results of kinetic experiments and quantum chemical computations on a series of platinum-promoted polycyclization reactions are described. Analyses of these results reveal a reactivity model that reaches beyond the energetics of the cascade itself, incorporating an ensemble of pre-cyclization conformations of the platinum-alkene reactant complex, only a subset of which are productive for bi- (or larger) cyclization and lead to products. Similarities and differences between this scenario, including reaction coordinates for polycyclization, for platinum- and enzyme-promoted polycyclization reactions are highlighted.

Mass Spectrometric Discrimination of Squalene Monohydroperoxide Isomers.

Squalene (SQ), a main component of human sebum, is readily photooxidized by exposure to sunlight, producing six squalene monohydroperoxide (SQ-OOH) isomers. Despite its known connection to various skin conditions, few studies have sought to analyze SQ-OOH at the isomeric level. In this study, we aimed to develop a method to discriminate each SQ-OOH isomer with the use of tandem mass spectrometry (MS/MS). The six standard SQ-OOH isomers were prepared by photooxidizing SQ in the presence of rose bengal, a photosensitizer, and isolated by semipreparative high-performance liquid chromatography (HPLC). To purify each isomer, 2-methoxypropene, which reversibly reacts with the hydroperoxide group of SQ-OOH, was utilized. Product ion scanning was then performed on the standard SQ-OOH isomers in the absence and presence of the sodium ion. In the absence of the sodium ion, the fragmentation patterns produced by atmospheric pressure chemical ionization were similar between the isomers, whereas in the presence of the sodium ion by electrospray ionization, unique fragmentation patterns were achieved. Based on these fragment ions, HPLC-MS/MS multiple reaction monitoring analysis was conducted on a mixture of the standard SQ-OOH isomers. We achieved discrimination of SQ-OOH isomers with high selectivity and detected SQ-OOH isomers at nanogram levels. These results may improve our understanding of the effect of SQ-OOH on skin conditions as well as the mechanism behind SQ peroxidation.

Directed evolution of squalene synthase for dehydrosqualene biosynthesis.

Squalene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a "dehydrosqualene synthase." All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.

Synthetic endeavours towards oxasqualenoid natural products containing 2,5-disubstituted tetrahydrofurans--eurylene and teurilene.

2,5-Disubstituted tetrahydrofurans constitute the core skeleton of several natural products and are pivotal synthetic analogues of medicinal importance that exhibit remarkable bioactivities. Oxasqualenoid natural products are implicated as potent biologically active molecules, particularly with regard to demonstrating significant cytotoxicity. Characteristic features of oxasqualenoids containing tetrahydrofuran fragments include the presence of a cis- and/or trans-2,5-disubstituted pattern in tetrahydrofuran moieties, and molecular symmetry is often noticed as well. Given their unique structural features combined with their bioactivity, two representative examples from this class of natural products, eurylene and teurilene, have been briefly reviewed. Eurylene, with reported cytotoxicity against lymphocytic leukemia, contains two non-adjacent linked cis- and trans-2,5-disubstituted tetrahydrofuran rings and a combined total of eight stereogenic centres. It is a chiral molecule due to the lack of a C2 axis of symmetry. Teurilene shows a prominent cytotoxicity on KB cells and has three adjacently linked 2,5-disubstituted tetrahydrofurans. A distinctive achiral facet is observed in teurilene, despite having eight stereocentres, due to the presence of meso symmetry (Cs). The prime objective of this account is to describe a precise mechanistic insight for both cis- and trans-2,5-disubstituted tetrahydrofurans present in these natural products and to highlight the exciting challenges encountered during the installation of functionalities or structural motifs en route to their synthetic approaches.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

Squalenoyl prodrug of paclitaxel: synthesis and evaluation of its incorporation in phospholipid bilayers.

1,1',2-Trisnorsqualenoic acid was conjugated to paclitaxel to obtain the squalenoyl-paclitaxel prodrug with the aim to improve the incorporation in phospholipid bilayers. Differential scanning calorimetry technique was employed to compare the interaction of squalenoyl-paclitaxel prodrug and free paclitaxel with phospholipid bilayers. The possibility of using lipid vesicles as carrier for the prodrug was also evaluated. An increased encapsulation into phospholipid bilayers of squalenoyl-paclitaxel with respect to the free drug was observed. The ability of lipid vesicles to retain the loaded prodrug was also observed which make this system to be considered as carrier for the prodrug.

Synthesis of n-squalenoyl cytarabine and evaluation of its affinity with phospholipid bilayers and monolayers.

Cytarabine (1-ß-D-arabinofuranosylcytosine, Ara-C), a pyrimidine nucleoside analogue, is an attractive therapeutic agent for the treatment of both acute and chronic myeloblastic leukemias. 1,1',2-tris-nor-Squalene acid (squaleneCOOH) has been conjugated to cytarabine with the formation of the squalenoyl-cytarabine prodrug, in order to improve the drug lipophilicity and, consequently, the affinity towards the environment of biological membranes, as well as of lipophilic carriers. The interaction of cytarabine and its prodrug with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles and monolayers has been studied by the differential scanning calorimetry and the Langmuir-Blodgett techniques. The interaction has been evaluated considering the effect of the compounds on the DMPC MLV and monolayers behaviour. The aim was to have information on the interaction of the drug and the prodrug with the biological membranes and on the possibility to use liposomes as carriers for the prodrug. The results showed an improved affinity of the prodrug with MLV and monolayers with respect to the free drug.

Novel pyrrole- and 1,2,3-triazole-based 2,3-oxidosqualene cyclase inhibitors.

Pyrrole- and 1,2,3-triazole-based 2,3-oxidosqualene cyclase (OSC) inhibitors 3 and 4 were discovered by conducting a virtual screening, a docking study based on the crystallographic structure of OSC, and biological assays. The hit rate of the assays was increased by establishing appropriate substructural filters in the virtual screening stage. Amide derivatives of 8 and 12 preserved the inhibitory activity of parent compound 3, which provided a reasonable starting point for further structure-activity-relationship (SAR) studies on related compounds.

A short total synthesis of (+)-omaezakianol via an epoxide-initiated cationic cascade reaction.

(+)-Omaezakianol was synthesized in only three steps from racemic chlorohydrin 4 by Shi epoxidation followed by cascade cyclization and reduction.

Stereo- and regioselective synthesis of squalene tetraepoxide.

Squalene tetraepoxide, a putative biosynthetic precursor to a variety of oxacyclic triterpenoid natural products, has been efficiently synthesized by anionic coupling of two farnesol-derived diepoxides, which have arisen from electronic control of the regioselectivity in organocatalytic enantioselective epoxidations.

Cyclization cascade of the C33-bisnorheptaprenoid catalyzed by recombinant squalene cyclase.

The enzymatic cyclization reaction of polyprenoid C(33) by squalene-hopene cyclase (SHC) was investigated with the intention of creating an unnatural hexacyclic compound. The enzymatic products consisted of mono-, bi-, tri-, tetra- and pentacyclic skeletons; however, hexacyclic products were not generated, contrary to our expectations. The absence of a hexacyclic skeleton indicated that the entire carbon chain of C(33) polyprene could not be included in the reaction cavity. Formation mechanisms of the products having mono- to pentacycles were discussed. Both chair/chair/boat conformation and chair/chair/chair conformations were formed for a tricycle, and both chair/chair/chair/boat conformation and chair/chair/chair/chair structures were constructed for a tetracycle. The pentacyclic product was created from the chair/chair/chair/chair/boat conformation. Squalene was folded in an all pre-chair conformation inside the reaction cavity to form the hopene skeleton. Therefore, the formation of a boat structure during the polycyclization reaction indicated that the molecule of polyprene C(33) was folded improperly due to incorrect arrangement/positioning in the reaction cavity. The creation of the hexacyclic core failed; however, it should be noted that SHC possessed great potential to tolerate the elongated squalene analog C(33), thus leading to the creation of novel compounds with C(33).

Total synthesis and determination of the absolute configuration of (+)-omaezakianol.

The proof of the pudding: The first asymmetric total synthesis of the marine tetracyclic oxasqualenoid (+)-omaezakianol features a convergent olefin cross-metathesis between a monotetrahydrofuran fragment and a triepoxy alkene, and cascade oxacyclizations of a triepoxy alcohol to form the right-hand three ether rings. The total synthesis proved the absolute configuration of (+)-omaezakianol to be that shown.

The role of chemical synthesis in structure elucidation of oxasqualenoids.

Recently, highly oxidized and structurally diverse triterpene polyethers, which are thought to be biogenetically squalene-derived natural products (oxasqualenoids), have been isolated from both marine and terrestrial organisms. However, it is often difficult to determine their stereostructures even by the current, highly advanced spectroscopic methods, especially in acyclic systems including stereogenic quaternary carbon centers. In such cases, it is effective to predict and synthesize the possible stereostructures. Herein, we report total assignments of the previously incomplete stereostructures of an epoxy tri-THF diol, intricatetraol and enshuol, members of the oxasqualenoids, through the first asymmetric total syntheses of the natural products, the configurations of which are difficult to determine by other means. Since this article is basically written as a communication without detailed experimental procedures and spectroscopic data, original papers with full data should follow.

Ion-trap tandem mass spectrometric analysis of squalene monohydroperoxide isomers in sunlight-exposed human skin.

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.

Mild TiIII- and Mn/ZrIV-catalytic reductive coupling of allylic halides: efficient synthesis of symmetric terpenes.

Two new efficient methods for the regioselective homocoupling of allylic halides using either catalytic TiIII or the combination Mn/ZrIV catalyst have been developed. The regio- and stereoselectivity of the process proved to increase significantly when the Mn/ZrIV catalyst is used as the coupling reagent and when cyclic substituted allylic halides are used as substrates. The use of Lewis acids such as collidine hydrochloride allowed the quantity of catalyst to be lowered up to 0.05 equiv. We have proved the utility of these protocols with the synthesis of different terpenoids such as (+)-beta-onoceradiene (1), (+)-beta-onocerine (2), squalene (5), and advanced key-intermediates in the syntheses of (+)-cymbodiacetal (3) and dimeric ent-kauranoids as xindongnin M (4a).

Application of the B-alkyl suzuki-miyaura cross-coupling reaction to the stereoselective synthesis of analogues of (3S)-oxidosqualene.

[reaction: see text] A general method is described for the direct and stereoselective synthesis of epoxypolyenes via Suzuki-Miyaura cross-coupling reaction of 1-iodoalkenes with B-alkylboron compounds. It allows for the straightforward and convergent assembly of compounds that are structurally similar to (3S)-oxidosqualene, an important intermediate in steroid biosynthesis.