Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

6-Methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone from Salvia aegyptiaca

From the whole plant of Salvia aegyptiaca, 6-methylcryptoacetalide, 6-methyl-epicryptoacetalide and 6-methylcryptotanshinone have been isolated and characterized, mainly by spectroscopic means. In addition to these novel diterpenoids, the known compounds 3beta-hydroxy-olean-12-en-28-oic acid, 3beta-hydroxy-oleana-11,13(18)-dien-28-oic acid, sitosterol-3beta-glucoside, sitosterol, stigmasterol, 5-hydroxy-7,3',4'-trimethoxyflavone and 5, 6-dihydroxy-7,3',4'-trimethoxyflavone were isolated.     

SYNTHESIS OF 6-FORMYLURIDINE 5′-MONOPHOSPHATE: 6-FORMYL-UMP

A synthesis of 6-formyluridine 5′-monophosphate (6-formylUMP) in 6 steps starting from uridine is described. This approach should be applicable to the preparation of other O5′-phosphorylated 6-formylUrds such as 6-formylUDP and 6-formylUTP.     

Synthesis and antifungal activity of 6-arylthio-/6-arylamino-4,7-dioxobenzothiazoles

6-Arylthio-/6-arylamino-4,7-dioxobenzothiazoles were synthesized and tested for in vitro antifungal activity against Candida species and Aspergillus niger. 6-Arylamino-4,7-dioxobenzothiazoles 5 and 6 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzothiazoles 3 and 4. The 6-arylamino-substituted compounds 5 and 6 exhibited the greatest activity. In contrast, 6-arylthio-, 2-/5-methyl- or 5-methoxy-moieties of compounds 3-4 did not improve their antifungal activity significantly. The results of this study suggest that 6-arylamino-4,7-dioxobenzothiazoles would be potent antifungal agents.     

Synthesis of kanamycin A analogs containing a 6-amino-6-deoxyglycofuranose moiety

Three kanamycin A analogs containing 6-amino-6-deoxyglycofuranoses have been prepared as candidates for potential activity against resistant bacteria producing 6'-N-acetyltransferase. They are 4-O-(6-amino-3,5,6-trideoxy-alpha-D-, -beta-D-, and -beta-L-erythro -hexofuranosyl)-6-O-(3-amino-3-deoxy-alpha-D-glucopyranosyl)-2,5-dideoxy-5-epi-5-fluorostreptamine. Structure-activity relationships of these compounds are discussed.     

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.     

The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex

Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.     

Synthesis and stereochemistry of 6-deoxy-6-halogeno-D-glucofuranose cyclic phosphates.

1, 2-O-Cyclohexylidene- and 1, 2-O-isopropylidene-alpha-D-glucofuranose react with hexa-alkylphosphorous triamides to give the corresponding 3, 5, 6-phosphites. Treatment of the latter compounds with chlorine and bromine affords 1, 2-substituted 6-deoxy-6-halogeno-alpha-D-glucofuranose 3, 5-phosphorohalogenidates. Replacement of halogen at phsophorus by hydroxyl and amino groups has been investigated. Cyclic phosphorohalogenidates isomerize in N, N-dimethylformamide. The stereochemistry of the compounds investigated was established by using 1H- and 13C-n.m.r. data.     

DNA-binding properties of Smc6, a core component of the Smc5-6 DNA repair complex

The Smc5-6 complex is an essential regulator of chromosome integrity and a key component of the DNA damage response. As an essential DNA repair factor, the Smc5-6 complex is expected to interact with DNA and/or chromatin during the execution of its functions. How the Smc6 protein promotes the binding of the Smc5-6 complex to DNA lesions is currently unknown. We show here that Smc6 is a strong DNA-binding protein with a clear preference for single-stranded DNA substrates. Importantly, Smc6 associates with DNA in the absence of other Smc5-6 complex components and its activity is modulated by nucleotides. Our results also show that the minimal size of single-stranded DNA required for tight association with Smc6 is ~60 nucleotides in length. Taken together, our results suggest that Smc6 contributes to DNA repair in vivo by targeting the Smc5-6 complex to single-stranded DNA substrates created during the processes of homologous recombination and/or DNA replication.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

KSGal6ST Is Essential for the 6-Sulfation of Galactose within Keratan Sulfate in Early Postnatal Brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

Synthesis of methyl 6'-deoxy-6'-fluoro-alpha-isomaltoside and of the corresponding trisaccharide

Methyl 6-O-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl)-2,3,4-tri- O-benzyl-alpha-D-glucopyranoside (5) was formed with high stereoselectivity when the condensation of methyl 2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl (1) with 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride in ether was promoted with silver perchlorate in the presence of 2,4,6-trimethylpyridine. O-Deacetylation of 5, followed by treatment of the formed 6, containing only HO-6' unsubstituted, with diethylaminosulfur trifluoride (DAST) or dimethylaminosulfur trifluoride (methyl DAST) gave the per-O-benzyl derivative (9) of methyl 6'-deoxy-6'-fluoro-alpha-isomaltoside. Compound 9 was also obtained by condensation of 1 with 2,3,4-tri-O-benzyl-6-deoxy-6-fluoro-beta-D-glucopyranosyl fluoride (4) in the presence of silver perchlorate and anhydrous stannous chloride. The fully benzylated methyl alpha-glycoside (15) of 6-deoxy-6-fluoro-isomaltotriose, was obtained by condensation of 6 with 4. Hydrogenolysis of 9 and 15 gave the methyl alpha-glycosides of isomaltose and isomaltotriose fluorinated at C-6 of their (nonreducing) D-glucosyl group. Fluoride-ion displacements involving DAST and methyl DAST gave practically identical results, but mixtures arising from reactions involving the latter reagent were lighter-colored and easier to resolve by chromatography. The isolation of methyl alpha-glycosides of 6'-deoxy-6'-fluorogentiobiose and of 6'-O-(6-deoxy-6-fluoro-beta-D-glucopyranosyl) isomaltose is also described.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines

In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].     

6-Bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives as novel, potent, and selective 5-HT6 receptor antagonists

A novel series of 6-bicyclopiperazinyl-1-arylsulfonylindoles and 6-bicyclopiperidinyl-1-arylsulfonylindoles derivatives was synthesized and found to be potent and selective 5-HT6 receptor antagonists.     

Stability of RNA hairpin loops: A 6 -C m -U 6

The thermodynamics and circular dichroism of a series of A₆-C_m-U₆ (m = 4, 5, 6 or 8) oligoribonucleotides have been studied. These molecules form intramolecular hairpin loops at low temperatures and therefore are useful models for similar structures which occur in larger, natural RNA molecules. The stability of the helix forming the stem of these loops was found to be considerably greater than an intermolecular helix with the same length and composition. The most stable loop is m = 6. The enthalpy for initiation of the loop is unfavorable; it ranges from + 24 kcal, for m = 4 to + 21 kcal, for m = 6. The maximum in stability for the C₆ loop and the large positive enthalpy for loop initiation are in disagreement with expectations from simple theories assuming a Gaussian distribution of end-to-end distances. Loop strain for m = 4 and m = 5 and the unstacking of the cytosines on loop formation are likely physical explanations for these thermodynamic data. The circular dichroism spectrum of cytosine residues in the C₆ and C₈ loops is very similar to the spectrum of single-stranded oligoribocytidylate. However, the cytosine residues in the C₅ loop have a very different circular dichroism spectrum from the corresponding oligo(C₅) spectrum. In accordance with the thermodynamic data, we conclude from the circular dichroism data that the C₅ loop has an altered conformation from the C₅ and C₈ loops.     

Interleukin-6

Autophagy reallocates nutrients and clears normal cells of damaged proteins and organelles. In the context of metastatic disease, invading cancer cells hijack autophagic processes to survive and adapt in the host microenvironment. We sought to understand how autophagy is regulated in the metastatic niche for prostate cancer (PCa) cells where bone marrow stromal cell (BMSC) paracrine signaling induces PCa neuroendocrine differentiation (NED). In PCa, this transdifferentiation of metastatic PCa cells to neuronal-like cells correlates with advanced disease. Because autophagy provides a survival advantage for cancer cells and promotes cell differentiation, we hypothesized that autophagy mediates PCa NED in the bone. Thus, we determined the ability of paracrine factors in conditioned media (CM) from two separate BMSC subtypes, HS5 and HS27a, to induce autophagy in C4-2 and C4-2B bone metastatic PCa cells by characterizing the autophagy marker, LC3. Unlike HS27a CM, HS5 CM induced LC3 accumulation in PCa cells, suggesting autophagy was induced and indicating that HS5 and HS27a secrete a different milieu of paracrine factors that influence PCa autophagy. We identified interleukin-6 (IL-6), a cytokine more highly expressed in HS5 cells than in HS27a cells, as a paracrine factor that regulates PCa autophagy. Pharmacological inhibition of STAT3 activity did not attenuate LC3 accumulation, implying that IL-6 regulates NED and autophagy through different pathways. Finally, chloroquine inhibition of autophagic flux blocked PCa NED; hence autophagic flux maintains NED. Our studies imply that autophagy is cytoprotective for PCa cells in the bone, thus targeting autophagy is a potential therapeutic strategy.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.