Straightforward access to novel mitochondriotropics derived from 2-arylethanol as potent and selective antiproliferative agents.

The necessity for developing novel cytostatic agents with improved activities and reduced side-effects to tackle cancer prompted us to investigate mitochondria-targeted compounds, an approach that is gaining attention for the selective transportation of cytotoxic agents. We envisioned the possibility of conjugating a phenethyl alcohol motif, decorated with a series of phenol-based substituents on the aryl moiety, with a triphenyl phosphonium scaffold (a mitochondria-directed vector), through a hydrocarbon chain of different lengths. Thus, such compounds that incorporate the phenethyl skeleton can be considered as masked phenolic compounds derived from relevant natural counterparts found in olive tree (e.g. tyrosol, hydroxytyrosol). Title compounds exhibited very strong in vitro antiproliferative activities against the panel of six human tumor cell lines tested, with GI50 values ranging from the nanomolar (0.026 ± 0.010 µM for 36) to the submicromolar range in most of the cases; this represents an improvement of up to 350-fold compared to classical chemotherapeutic agents, like 5-fluorouracil or cisplatin. Interestingly, decrease in the linker length led to an increase of GI50 values against non-tumor cells, thus allowing a remarkable improvement of selectivity (SI up to 269). The very promising antiproliferative activities prompted us to further investigate their behaviour against multidrug resistant cell lines (MDR). The results indicated a reduced sensitivity of the multidrug resistant cells to compounds, probably due to P-gp-mediated efflux of these antiproliferative agents. Interestingly, activities were completely restored to the same levels by co-administration of tariquidar, a well-known inhibitor of P-gp. Flow cytometry analysis on sensitive cell lines revealed a decrease in the percentage of cells in G1 phase accompanied by increase in S and G2/M phases. In addition, a significant increase in subG1 area, was observed. These results are compatible with the necrotic and apoptotic cell death detected in the Annexin V assay, and with the depolarization of the mitochondria membrane. Thus, the new mitochondriotropic agents reported herein can be considered as promising antiproliferative agents, endowed with remarkable potency and selectivity, including MDR cells, upon co-administration with a pump-efflux inhibitor.

A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Direct transfer of tri- and di-fluoroethanol units enabled by radical activation of organosilicon reagents.

Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z'.

Avertin affects murine colitis by regulating neutrophils and macrophages.

Anesthetics are thought to be involved in immunomodulation. Avertin is one of the safest and most commonly used intravenous anesthetics in rodent experiments; it is also widely used in euthanasia of inflammatory bowel disease (IBD) models. This study aimed to define the role and mechanism of action of Avertin on murine colitis. We assessed the effects of a single Avertin injection on colitis using the disease activity index (DAI), pathology, enzyme-linked immunosorbent assay (ELISA), multiplex-ELISA, flow cytometry, and routine blood examination in wild-type (WT) and dextran sodium sulphate (DSS)-treated mice. Although Avertin caused acute cecitis in WT mice after 24 h and aggravated inflammation in the medium term, it alleviated inflammation in the late stage of DSS-induced colitis according to the DAI. Avertin upregulated MPO production and induced the accumulation of neutrophils and macrophages in intestinal mucosa of both WT and DSS-treated mice; the altered MPO might indicate a change in respiratory burst. However, it exhibited a more effective suppression of inflammatory factors secreted by macrophages as the colitis progressed. Avertin led to an increase in neutrophils and decrease in monocytes in both WT and DSS-treated mice blood. Our findings suggest that Avertin aggravates inflammation in the early and medium terms, but alleviates inflammation in the late stage of colitis by regulating neutrophils and macrophages.

Poly(2-oxazoline)s with a 2,2'-Iminodiacetate End Group Inhibit and Stabilize Laccase.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.

Environmental impact of combustion of ethanolic biodiesel/diesel blends from several feedstocks on the gas emission levels in the atmosphere.

The aim of this work was to evaluate simultaneously the effect of produced ethanolic biodiesel from several feedstocks (soybean, crambe, macaw, sunflower, and waste cooking oil) and engine operational conditions (low and high engine speed) during combustion of biodiesel/diesel blends on the N2O, NOx, NO, CO2, and CO emission levels in the atmosphere. The biodiesel samples were prepared in one and/or two reaction steps, according to the acid index of each raw material, by esterification using H2SO4 and/or chemical transesterification using sodium ethoxide, both, through ethanolic route. The quality of the produced biodiesels was confirmed by ASTM/EN specifications. Then, biodiesel/diesel blends were prepared according to the following proportions: 10% (B10), 15% (B15), 25% (B25), and 50% (B50). In general way, all raw materials under combustion at low and high engine speed contributed to the formation of NOx and this effect was more drastically increased as the biodiesel concentration in the blends increases. N2O presented a similar behavior except for blends containing crambe and macaw biodiesel whose emissions were slightly reduced as a function of biodiesel content in these blends. Then, Principal component analysis (PCA) was applied to discriminate the effect of engine operating conditions, biodiesel kind, and biodiesel content in the blends during their combustion on the exhaust emissions. The attained results point to crambe and macaw as more environmentally sustainable feedstocks for biodiesel production because they generate less greenhouse gas emissions. These results are particularly attractive considering that, both, crambe and macaw are non-edible feedstocks with great potential for biodiesel production.

Cloning, expression, purification, and kinetic characterization of mitochondrial thioredoxin (TsTrx2), cytosolic thioredoxin (TsTrx1), and glutaredoxin (TsGrx1) from Taenia solium.

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.

The effect of avertin anesthesia and a mixture of three types of anesthetic agents on food intakeand body weight in high fat-induced obese maleand female mice.

Basic research on obesity is becoming more important due to an increasing number of obese people. Experiments using obesity-model animals often require surgical interventions, such as gastric operation, and proper selection of anesthesia is important. Avertin, an agent mainly composed of 2,2,2-Tribromoethanol, has been used as general anesthesia for a long time, without the use of narcotic drugs. In the current study, we found that a single injection of avertin can decrease body weight (BW) in male and female C57BL/6J and ICR mice with high fat-diet (HFD)-induced obesity, but not in standard diet-fed nonobese males and females. Because the BW-reducing effect was more prominent in the female mice, we compared the effects of avertin and a mixture of three types of anesthetic agents (3MIX), which was developed in 2011, on BW reduction in HFD-induced obese female mice. Although both avertin and 3MIX decreased food intake and BW, the effects of avertin were significantly more potent than those of 3MIX. C-Fos expression, a neural activation marker, was dramatically increased in the brain regions related to the regulation of both food intake and the autonomic nervous system after avertin injection, but not after 3MIX injection. This suggests that avertin strongly stimulates the center of feeding regulation and the autonomic nervous system and therefore decreases BW. The current study suggests the advantages of using 3MIX for surgical interventions in mice in obesity research, as it is ideal to prevent anesthesia-induced BW decline.

Kinetic characterization of wild-type and mutant human thioredoxin glutathione reductase defines its reaction and regulatory mechanisms.

In most cells, the thioredoxin (Trx) and glutathione systems are essential in maintaining redox homeostasis. The selenoprotein thioredoxin glutathione reductase (TGR) is a hybrid enzyme in which a glutaredoxin (Grx) domain is linked to a thioredoxin reductase (TrxR). Notably, the protein is also capable of reducing glutathione disulfide (GSSG), thus representing an important link between the two redox systems. In this study, we recombinantly produced human TGR (hTGR wild-type) by fusing its open reading frame with a bacterial selenocysteine insertion sequence element and co-expressing the construct in Escherichia coli together with the selA, selB, and selC genes. Additionally, the Sec→Cys mutant (hTGRU642C ) of the full-length protein, the isolated TrxR domain (hTGR151-643 ) and the Grx domain containing a monothiol active site (hTGR1-150 ) were produced and purified. All four proteins were kinetically characterized in direct comparison using Trx, DTNB, HED, or GSSG as the oxidizing substrate. Interestingly, the HED reduction activity was Sec independent and comparable in the full-length protein and the isolated Grx domain, whereas the TrxR and glutathione reductase reactions were clearly selenocysteine dependent, with the GR reaction requiring the Grx domain. Site-directed mutagenesis studies revealed novel insights into the mechanism of GSSG reduction. Furthermore, we identified several glutathionylation sites in hTGR, including Cys93, Cys133, and Cys619, and an inhibitory effect of these modifications on enzyme activity. In contrast to other TGRs, for example, from platyhelminth parasites, hTGR did not exhibit hysteretic behavior. These findings provide new insights into the reaction mechanism and regulation of monothiol Grx-containing TGRs. DATABASE: EC numbers: 1.8.1.9; 1.8.1.B1.

2-18F-Fluoroethanol Is a PET Reporter of Solid Tumor Perfusion.

Solid tumor perfusion is a proven variable of interest for predicting cancer aggression and response to therapy. Current methods for noninvasively imaging tumor perfusion with PET are limited by restricted accessibility and short half-lives of perfusion radiotracers. This study presents 2-18F-fluoroethanol (2-18F-FEtOH) as a perfusion reporter that can distinguish between tumors of varying perfusion levels and can be applied to screening drugs that modify tumor perfusion. Methods: Uptake of 2-18F-FEtOH in 4T1 and 67NR murine mammary carcinoma tumors grown in mice was measured using ex vivo radiography as well as static and dynamic PET imaging. 2-18F-FEtOH uptake was directly compared with the 14C-iodoantipyrine perfusion reporter, and the perfusion-modifying drugs nicotinamide, pentoxifylline, and hydralazine were used to manipulate tumor perfusion before 2-18F-FEtOH quantification. Results: Uptake of 2-18F-FEtOH in 4T1 and 67NR tumors was consistent with known perfusion differences within and between these tumors. 2-18F-FEtOH uptake corresponded well with 14C-iodoantipyrine and reflected the tumor perfusion-modifying effects of each drug. Conclusion: 2-18F-FEtOH is a novel 18F-based radiotracer for investigating tumor perfusion with PET imaging. Quantification of 2-18F-FEtOH uptake can be used to distinguish between tumors of varying perfusion and to screen the efficacy of blood flow-modifying drugs for use as adjuvants to existing cancer therapies.

Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol.

(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 µmol/min/mg, ten times of that of the wild-type. Its k cat/K m increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.

Refinements for embryo implantation surgery in the mouse: comparison of injectable and inhalant anesthesias - tribromoethanol, ketamine and isoflurane - on pregnancy and pup survival.

An essential aspect of genetically-engineered mice (GEM) is the ability to produce live animals after the appropriate injection procedure. Animals are produced by implantation of manipulated embryos into pseudopregnant females for gestation, parturition, and growth to the weaning stage. This study was carried out to test whether the anesthesia used during surgery could affect the number of pups produced. Anesthetics commonly used for implant surgery include tribromoethanol (Avertin) delivered by intraperitoneal (IP) injection, IP-injected ketamine:xylazine or ketamine:medetomidine mix, and inhaled isoflurane. To determine if the anesthesia used might affect the number of animals produced, each anesthetic agent was tested in implant surgeries and the numbers of pups produced using both wild-type and GEM embryos were assessed. Parallel studies were conducted in institutions in the EU and in the USA. Based on a direct comparison of pregnancy status, number of pups born, and number of pups weaned for each agent, we found no statistical differences among the three anesthetics. We conclude that all three anesthetic agents tested are equally useful for implantation surgery.

Triazole analog 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol induces reactive oxygen species and autophagy-dependent apoptosis in both in vitro and in vivo breast cancer models.

Autophagy is considered as an important cell death mechanism that closely interacts with other common cell death programs like apoptosis. Critical role of autophagy in cell death makes it a promising, yet challenging therapeutic target for cancer. We identified a series of 1,2,3-triazole analogs having significant breast cancer inhibition property. Therefore, we attempted to study whether autophagy and apoptosis were involved in the process of cancer cell inhibition. The lead molecule, 1-(1-benzyl-5-(4-chlorophenyl)-1H-1,2,3-triazol-4-yl)-2-(4-bromophenylamino)-1-(4-chlorophenyl)ethanol (T-12) induced significant cell cycle arrest, mitochondrial membrane depolarization, apoptosis and autophagy in MCF-7 and MDA-MB-231 cells. T-12 increased reactive oxygen species and its inhibition by N-acetyl-L-cysteine protected breast cancer cells from autophagy and apoptosis. Autophagy inhibitor, 3-methyladenine abolished T-12 induced apoptosis, mitochondrial membrane depolarization and reactive oxygen species generation. This suggested that T-12 induced autophagy facilitated cell death rather than cell survival. Pan-caspase inhibition did not abrogate T-12 induced autophagy, suggesting that autophagy precedes apoptosis. In addition, T-12 inhibited cell survival pathway signaling proteins, Akt, mTOR and Erk1/2. T-12 also induced significant regression of tumor with oral dose of as low as 10mg/kg bodyweight in rat mammary tumor model without any apparent toxicity. In presence of reactive oxygen species inhibitor (N-acetyl-L-cysteine) and autophagy inhibitor (chloroquine), T-12 induced tumor regression was significantly decreased. In conclusion, T-12 is a potent inducer of autophagy-dependent apoptosis in breast cancer cells both in vitro and in vivo and can serve as an important lead in development of new anti-tumor therapy.

Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques.

We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 µm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.

Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical 'one-way' active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.

Preparation of pharmaceutical important fluorinated 1-arylethanols using isolated enzymes.

Fluorinated 1-arylethanols are important building blocks in medicinal chemistry especially for preparing kinase inhibitors for cancer therapy, NK1 receptor antagonists and drugs used in treatment of osteoporosis. Asymmetric reduction of carbonyl groups using enzymes is one of the key technologies to obtain such molecules in enantiomerically pure form. By using isolated enzymes coupled with cofactor recycling, highly concentrated, robust and economical processes can be developed. The aim of this review is to give an overview of biocatalytic carbonyl reduction with special focus on processing of fluoro containing 1-arylethanones with enzymes in cell free systems. The methodologies applied to improve the reactions are highlighted, alongside potential application of the building blocks in bioactive compounds. Enzymatic ketone reduction is concluded to be most beneficial as compared to chemo catalytic methods in transformations of highly fluorinated and therefore electron deficient ketones. A high enantiomeric excess can be achieved, and by changing the catalyst, both enantiomers are accessible. High rates are often seen for such electron deficient ketones, and the reactions have a favourable equilibrium position.

f-[18F]fluoroethanol and 3-[18F]fluoropropanol: facile preparation, biodistribution in mice, and their application as nucleophiles in the synthesis of [18F]fluoroalkyl aryl ester and ether PET tracers.

INTRODUCTION: 2-[(18)F]Fluoroethoxy and 3-[(18)F]fluoropropoxy groups are common moieties in the structures of radiotracers used with positron emission tomography. The objectives of this study were (1) to develop an efficient one-step method for the preparation of 2-[(18)F]fluoroethanol (2-[(18)F]FEtOH) and 3-[(18)F]fluoropropanol (3-[(18)F]FPrOH); (2) to demonstrate the feasibility of using 2-[(18)F]FEtOH as a nucleophile for the synthesis of 2-[(18)F]fluoroethyl aryl esters and ethers; and (3) to determine the biodistribution profiles of 2-[(18)F]FEtOH and 3-[(18)F]FPrOH in mice. METHODS: 2-[(18)F]FEtOH and 3-[(18)F]FPrOH were prepared by reacting n-Bu4N[(18)F]F with ethylene carbonate and 1,3-dioxan-2-one, respectively, in diethylene glycol at 165°C and purified by distillation. 2-[(18)F]fluoroethyl 4-fluorobenzoate and 1-(2-[(18)F]fluoroethoxy)-4-nitrobenzene were prepared by coupling 2-[(18)F]FEtOH with 4-fluorobenzoyl chloride and 1-fluoro-4-nitrobenzene, respectively. Biodistribution and PET/CT imaging studies of 2-[(18)F]FEtOH and 3-[(18)F]FPrOH were performed in normal female Balb/C mice. RESULTS: The preparation of 2-[(18)F]FEtOH and 3-[(18)F]FPrOH took 60 min, and their decay-corrected yields were 88.6 ± 2.0% (n = 9) and 65.6 ± 10.2% (n = 5), respectively. The decay-corrected yields for the preparation of 2-[(18)F]fluoroethyl 4-fluorobenzoate and 1-(2-[(18)F]fluoroethoxy)-4-nitrobenzene were 36.1 ± 5.4% (n = 3) and 27.7 ± 10.7% (n = 3), respectively. Imaging/biodistribution studies in mice using 2-[(18)F]FEtOH showed high initial radioactivity accumulation in all major organs followed by very slow clearance. On the contrary, by using 3-[(18)F]FPrOH, radioactivity accumulated in all major organs was cleared rapidly, but massive in vivo defluorination (31.3 ± 9.57%ID/g in bone at 1h post-injection) was observed. CONCLUSIONS: Using 2-[(18)F]FEtOH/3-[(18)F]FPrOH as a nucleophile is a competitive new strategy for the synthesis of 2-[(18)F]fluoroethyl/3-[(18)F]fluoropropyl aryl esters and ethers. Our biodistribution data emphasize the importance of in vivo stability of PET tracers containing a 2-[(18)F]fluoroethyl or 3-[(18)F]fluoropropyl group due to high background and high bone uptake resulting from 2-[(18)F]FEtOH and 3-[(18)F]FPrOH, respectively. This is especially important for their aryl ester derivatives which are prone to in vivo hydrolysis.

Photoresponsive cross-linked polymeric particles for phototriggered burst release.

We synthesized a series of cross-linked photoresponsive polymeric particles with photolabile monomers and cross-linkers through miniemulsion polymerization. These particles are quite stable in dark, while light irradiation caused the breakage of particles and the efficient release of encapsulated contents up to 95% based on Nile red fluorescence. Photoswitches of particle systems were confirmed by fluorescence spectroscopy, SEM and colorimetry. Particle uptake and triggered release in RAW264.7 cells were confirmed by fluorescein diacetate loaded particles.

A bioactive probe of the oxidative pentose phosphate cycle: novel strategy to reverse radioresistance in glucose deprived human colon cancer cells.

The specific effects of glucose deprivation on oxidative pentose phosphate cycle (OPPC) function, thiol homeostasis, protein function and cell survival remain unclear due to lack of a glucose-sensitive chemical probe. Using p53 wild type and mutant human colon cells, we determined the effects of hydroxyethyl disulfide (HEDS) on NADPH, GSH, GSSG, total glutathione, total non-protein and protein thiol levels, the function of the DNA repair protein Ku, and the susceptibility to radiation-induced free radicals under normal glucose or glucose-deprived conditions. HEDS is rapidly detoxified in normal glucose but triggered a p53-independent metabolic stress in glucose depleted state that caused loss of NADPH, protein and non-protein thiol homeostasis and Ku function, and enhanced sensitivity of both p53 wild type and mutant cells to radiation induced oxidative stress. Additionally, high concentration of HEDS alone induced cell death in p53 wild type cells without significant effect on p53 mutant cells. HEDS offers a useful tool to gain insights into how glucose metabolism affects OPPC dependent stress-induced cellular functions and injury, including in tumor cells, where our findings imply a novel therapeutic approach to target glucose deprived tumor. Our work introduces a novel probe to address cancer metabolism and ischemic pathology.

Hydroxyethyl disulfide as an efficient metabolic assay for cell viability in vitro.

Cell viability assays have a variety of well known practical and technical limitations. All the available approaches have disadvantages, such as non-linearity, high background and cumbersome protocols. Several commonly used tetrazolium chemicals rely upon generation of a colored formazan product formed by mitochondrial reduction of these compounds via phenazine methosulfate (PMS). However, sensitivity is inherently limited because their reduction relies on mitochondrial bioreduction and cellular transport of PMS, as well as accessibility to tetrazolium chemicals. In this study, we identify hydroxethyldisulfide (HEDS) as an inexpensive probe that can measure cellular metabolic activity without the need of PMS. In tissue culture medium, HEDS accurately quantitated metabolically active live cells in a linear manner superior to tetrazolium based and other assays. Cell toxicity produced by chemotherapeutics (cisplatin, etoposide), oxidants (hydrogen peroxide, acetaminophen), toxins (phenyl arsine oxide, arsenite) or ionizing radiation was rapidly determined by the HEDS assay. We found that HEDS was superior to other commonly used assays for cell viability determinations in its solubility, membrane permeability, and intracellular conversion to a metabolic reporter that is readily transported into the extracellular medium. Our findings establish the use of HEDS in a simple, rapid and low cost assay to accurately quantify viable cells.