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Improvements in the synthesis of carbon-linked glucuronide/glucoside conjugates of cancer chemopreventive retinoids have been achieved starting with 2,3,4,6-tetra-O-benzyl-D-glucopyranose. The revised approach demonstrates better yields, eliminates the use of an expensive, carcinogenic protecting group reagent, and avoids much painstaking chromatography. The new approach should allow synthesis of larger quantities of the agents for detailed animal and mechanistic studies. [ABSTRACT FROM PUBLISHER]

Retinoid X receptor (RXRα) is activated by 9-cis-retinoic acid (9cRA) and regulates transcription as a homodimer or as a heterodimer with other nuclear receptors. We have previously demonstrated that β-apo-13-carotenone, an eccentric cleavage product of α-carotene, antagonizes the activation of RXRα by 9cRA in mammalian cells overexpressing this receptor. However, the molecular mechanism of β-apo-13-carotenone's modulation on the transcriptional activity of RXRαis not understood and is the subject of this report. We performed transactivation assays using full-length RXRαand reporter gene constructs (RXRE-Luc) transfected into COS-7 cells, and luciferase activity was examined. β-apo-13-carotenone was compared with the RXRα antagonist UVI3003. The results showed that both β-apo-13-carotenone and UVI3003 shifted the dose-dependent RXRαactivation by 9cRA. In contrast, the results of assays using a hybrid Gal4-DBD:RXRαLBD receptor reporter cell assay that detects 9cRA-induced coactivator binding to the ligand binding domain demonstrated that UVI3003 significantly inhibited 9cRA-induced coactivator binding to RXRαLBD, but β-apo-13-carotenone did not. However, both β-apo-13-carotenone and UVI3003 inhibited 9-cRA induction of caspase 9 gene expression in the mammary carcinoma cell line MCF-7.Toresolve this apparent contradiction, we investigated the effect of β-apo-13-carotenone on the oligomeric state of purified recombinant RXRαLBD. β-apo-13-carotenone induces tetramerization of the RXRαLBD, although UVI3003 had no effect on the oligomeric state. These observations suggest that β-apo-13-carotenoneregulates RXR αtranscriptional activity by inducing the formation of the "transcriptionally silent" RXRαtetramer. [ABSTRACT FROM AUTHOR]

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15'-oxygenase (BCO1) catalyzes the oxidative cleavage of provitaminAcarotenoids at the central 15-15' double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (kcat/Km). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a Vmax=197.2 nmol retinal/mg BCO1×h, Km=17.2 μM and catalytic efficiency kcat/Km = 6098 M -1 min-1. The enzyme also catalyzed the oxidative cleavage of β-carotene, β-cryptoxanthin, and β-apo-8'-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of α-carotene. Surprisingly,BCO1catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10'-carotenal, β-apo-12'-carotenal, β-apo-14'-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies. [ABSTRACT FROM AUTHOR]

This article reports the CuCN-mediated O-alkylation of formamide with 2-bromomethylindole. In addition, the formyloxylation products have been successfully exploited in the synthesis of novel indol-2-ylmethyl ether derivatives. [ABSTRACT FROM AUTHOR]

Regiospecific chlorination of xylenes has been developed by employing NCS as a reagent and K-10 montmorrillonite clay as a solid support. [ABSTRACT FROM AUTHOR]

Abstract: 2-Aryl-1,3-indanedione and phthalide derivatives have attracted considerable interest due to their anticoagulant, parasiticidal and a range of biological activities. The synthesis of 2-aryl-1,3-indanedione by the condensation of the title sulfone with aryl aldehydes through an interesting pathway and a previously unreported approach to 3-alkylphthalide from C-alkylated derivatives of 1 under microwave and conventional heating conditions are described. [Copyright &y& Elsevier]

Abstract: Five novel 3-carboranyl thymidine analogues (3CTAs) were designed and synthesized for boron neutron capture therapy (BNCT) of cancer. Phosphorylation of all five 3CTAs was catalyzed by recombinant human thymidine kinase (hTK1) using adenosine triphosphate (ATP) as the phosphate donor. The obtained phosphorylation rates ranged from 4% to 64.5% relative to that of thymidine. The compound with the most favorable hTK1 binding properties had a k cat/K M value of 57.4% relative to that of thymidine and an IC50 of inhibition of thymidine phosphorylation by hTK1 of 92μM. Among the five synthesized 3CTAs, this agent had also the overall most favorable physicochemical properties. Therefore, it may have the potential to replace N5–2OH, the current lead 3CTA, in preclinical studies. An in silico model for the binding of this compound to hTK1 was developed. [Copyright &y& Elsevier]

Abstract: Texaphyrin macrocycles that contain gadolinium or lutetium, such as motexafin gadolinium and motexafin lutetium, are versatile anticancer therapeutics and diagnostics. Gadolinium texaphyrins substituted with carborane clusters could also find application in combined gadolinium and boron neutron capture therapy (GdB-NCT). The synthesis and characterization of novel texaphyrins containing gadolinium or lutetium in the pentaaza core and two carborane clusters bound to opposite pyrrol units of the macrocycle are described. [Copyright &y& Elsevier]

Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15β-dioxygenase (BCO1) at the central 15-15β double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9β,10β-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9β-10β bond to yield an ionone and an apo-10β-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chickenBCO2in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitaminAcarotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitaminAcarotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10β-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8β-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9β-10β bond to produce apo-10β-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this. [ABSTRACT FROM AUTHOR]

β-Carotene 15-15'-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase, incorporating oxygen from O2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-carotene in either 16O2-H218O or 18O2-H216O medium for 15 min at 37 °C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry. At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from 18O-retinal-H216O and 16O-retinal-H218O incubations to account for nonenzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase. [ABSTRACT FROM AUTHOR]

Enzymatic cleavage of the nonsymmetric provitamin A carotenoid α-carotene results in one molecule of retinal (vitamin A), and one molecule of α-retinal, a biologically inactive analog of true vitamin A. Due to structural similarities, α-retinyl esters and vitamin A esters typically coelute, resulting in the overestimation of vitamin A originating from α-carotene. Herein, we present a set of tools to identify and separate α-retinol products from vitamin A. α-Retinyl palmitate (αRP) standard was synthesized from α-ionone following a Wittig-Horner approach. A high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method employing a C30 column was then developed to separate the species. Authentic standards of retinyl esters and the synthesized α-RP confirmed respective identities, while other α-retinyl esters (i.e. myristate, linoleate, oleate, and stearate) were evidenced by their pseudomolecular ions observed in electrospray ionization (ESI) mode, fragmentation, and elution order. For quantitation, an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode was used, and retinol, the predominant in-source parent ion was selected and fragmented. The application of this method to a chylomicron-rich fraction of human plasma is demonstrated. This method can be used to better determine the quantity of vitamin A derived from foods containing α-carotene. [ABSTRACT FROM AUTHOR]

β-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, β-Carotene 15,15′-oxygenase (BCO1) converts β-Carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitaminAthat acts as a transcription factor ligand to regulate gene expression. β-Carotene can also be cleaved by β-Carotene 9′,10′-oxygenase (BCO2) to form β-apo-10′-carotenal, a precursor of retinoic acid and a transcriptional regulator per se. The mammalian embryo obtains β-Carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that β-Carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show thatβ-Carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that β-apo-10′-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of β-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that β-Carotene induces a feed-forward mechanism in the placenta to enhance the assimilation ofβ-Carotene for proper embryogenesis. [ABSTRACT FROM AUTHOR]

β-Apo-carotenoids, including β-apo-13-carotenone and β-apo-14′-carotenal, are potent retinoic acid receptor (RAR) antagonists in transactivation assays. We asked how these influence RAR-dependent processes in living cells. Initially, we explored the effects of β-apo-13-carotenone and β-apo-14′-carotenal on P19 cells, a mouse embryonal carcinoma cell line that differentiates into neurons when treated with all- trans -retinoic acid. Treatment of P19 cells with either compound failed to block all- trans -retinoic acid induced differentiation. Liquid chromatography tandem mass spectrometry studies, however, established that neither of these β-apo-carotenoids accumulates in P19 cells. All- trans -retinoic acid accumulated to high levels in P19 cells. This suggests that the uptake and metabolism of β-apo-carotenoids by some cells does not involve the same processes used for retinoids and that these may be cell type specific. We also investigated the effects of two β-apo-carotenoids on 3T3-L1 adipocyte marker gene expression during adipocyte differentiation. Treatment of 3T3-L1 adipocytes with either β-apo-13-carotenone or β-apo-10′-carotenoic acid, which lacks RAR antagonist activity, stimulated adipocyte marker gene expression. Neither blocked the inhibitory effects of a relatively large dose of exogenous all- trans -retinoic acid on adipocyte differentiation. Our data suggest that in addition to acting as transcriptional antagonists, some β-apo-carotenoids act through other mechanisms to influence 3T3-L1 adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Dietary carote-noids like β-carotene are converted within the body either to retinoid, via β-carotene-15,15′-dioxygenase (BCO1), or to β-apo-carotenoids, via β-carotene-9′,10′-oxygenase 2. Some β-apo-carotenoids are po-tent antagonists of retinoic acid receptor (RAR)-mediated transcrip-tional regulation, which is required to ensure normal heart develop-ment and functions. We established liquid chromatography tandem mass spectrometery methods for measuring concentrations of 10 β-apo-carotenoids in mouse plasma, liver, and heart and assessed how these are influenced by Bco1 deficiency and β-carotene intake. Sur-prisingly, Bco1-/- mice had an increase in heart levels of retinol, nonesterified fatty acids, and ceramides and a decrease in heart triglycerides. These lipid changes were accompanied by elevations in levels of genes important to retinoid metabolism, specifically retinol dehydrogenase 10 and retinol-binding protein 4, as well as genes involved in lipid metabolism, including peroxisome proliferator-acti-vated receptor-7, lipoprotein lipase, Cd36, stearoyl-CoA desaturase 1, and fatty acid synthase. We also obtained evidence of compromised heart function, as assessed by two-dimensional echocardiography, in Bco1-/- mice. However, the total absence of Bco1 did not substan-tially affect β-apo-carotenoid concentrations in the heart. β-Carotene administration to matched Bco1-/- and wild-type mice elevated total β-apo-carotenal levels in the heart, liver, and plasma and total β-apo-carotenoic acid levels in the liver. Thus, BCO1 modulates heart metabolism and function, possibly by altering levels of cofactors required for the actions of nuclear hormone receptors. [ABSTRACT FROM AUTHOR]

β-Carotene is the major dietary source of provitamin A. Central cleavage of β-carotene catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of β-carotene at noncentral double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β-apocarotenones, whose biological functions in mammals are unknown.Weused reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs. Importantly, however, β-apo-14′-carotenal, β-apo-14′- carotenoic acid, and β-apo-13-carotenone antagonized ATRAinduced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14′-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nM β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to evaluate the effectiveness of a 3-carboranyl thymidine analogue (3CTA), 3-[5-(2-(2,3-dihydroxyprop-1-yl)-o-carboran-1-yl)pentan-1-yl] thymidine, drsignated N5-20H, for boron neutron capture therapy (BNCT) of brain tumors using the RG2 rat glioma model. Target validation was established using the thymidine kinase (TK) 1(+) wild-type, murine L929 cell line and its TK1(-) mutant counterpart, which were implanted s.c. (s.c.) into nude mice. Two intratumoral (i.t.) injections of [sup10]B-enriched N5-20H were administered to tumor-bearing mice at 2-hour intervals, after which BNCT was carried out at the Massachusetts Institute of Technology (MIT) Research Reactor. Thirty days after BNCT, mice bearing TK1(+) L929 tumors had a 15x reduction in tumor volume compared with TK1(-) controls. Based on these favorable results, BNCT studies were then initiated in rats bearing intracerebral (i.c.) RG2 gliomas, after i.c. administrati4n of N5-20H by Alzet osmotic pumps, either alone or in combination with i.v. (i.v.) boronophenylalanine (BPA), a drug that has been used clinically. The mean survival times (MST5) of RG2 gli~oma bearing rats were 45.6 ± 7.2 days, 35.0 ± 3.3days, and 52.9 ± 8.9 days, respectively, for animals that received N5-20H, BPA, or both. The differences between the survival plots of rats that received N5-20H and BPA alone were highly significant (P = 0.0003). These data provide proof-of-principle that a 3CTA can function as a boron delivery agent for NCT. Further studies are planned to design and synthesize 3CTAs with enhanced chemical and biological properties, and increased therapeutic efficacy. [ABSTRACT FROM AUTHOR]

Concise synthetic methods for synthesizing 3-carboranyl thymidine analogues (3CTAs) modified with cyclic and acyclic alcohols have been developed. The synthesis of these potential boron neutron capture therapy (BNCT) agents and their preliminary biological evaluation is described. [ABSTRACT FROM AUTHOR]