Spatial and temporal dynamics of HDACs class IIa following mild traumatic brain injury in adult rats.

The fundamental role of epigenetic regulatory mechanisms involved in neuroplasticity and adaptive responses to traumatic brain injury (TBI) is gaining increased recognition. TBI-induced neurodegeneration is associated with several changes in the expression-activity of various epigenetic regulatory enzymes, including histone deacetylases (HDACs). In this study, PET/CT with 6-([18F]trifluoroacetamido)-1- hexanoicanilide ([18F]TFAHA) to image spatial and temporal dynamics of HDACs class IIa expression-activity in brains of adult rats subjected to a weight drop model of diffuse, non-penetrating, mild traumatic brain injury (mTBI). The mTBI model was validated by histopathological and immunohistochemical analyses of brain tissue sections for localization and magnitude of expression of heat-shock protein-70 kDa (HSP70), amyloid precursor protein (APP), cannabinoid receptor-2 (CB2), ionized calcium-binding adapter protein-1 (IBA1), histone deacetylase-4 and -5 (HDAC4 and HDAC5). In comparison to baseline, the expression-activities of HDAC4 and HDAC5 were downregulated in the hippocampus, nucleus accumbens, peri-3rd ventricular part of the thalamus, and substantia nigra at 1-3 days post mTBI, and remained low at 7-8 days post mTBI. Reduced levels of HDAC4 and HDAC5 expression observed in neurons of these brain regions post mTBI were associated with the reduced nuclear and neuropil levels of HDAC4 and HDAC5 with the shift to perinuclear localization of these enzymes. These results support the rationale for the development of therapeutic strategies to upregulate expression-activity of HDACs class IIa post-TBI. PET/CT (MRI) with [18F]TFAHA can facilitate the development and clinical translation of unique therapeutic approaches to upregulate the expression and activity of HDACs class IIa enzymes in the brain after TBI.

Succinate prodrugs as treatment for acute metabolic crisis during fluoroacetate intoxication in the rat.

Sodium fluoroacetate (FA) is a metabolic poison that systemically inhibits the tricarboxylic acid (TCA) cycle, causing energy deficiency and ultimately multi-organ failure. It poses a significant threat to society because of its high toxicity, potential use as a chemical weapon and lack of effective antidotal therapy. In this study, we investigated cell-permeable succinate prodrugs as potential treatment for acute FA intoxication. We hypothesized that succinate prodrugs would bypass FA-induced mitochondrial dysfunction, provide metabolic support, and prevent metabolic crisis during acute FA intoxication. To test this hypothesis, rats were exposed to FA (0.75 mg/kg) and treated with the succinate prodrug candidate NV354. Treatment efficacy was evaluated based on cardiac and cerebral mitochondrial respiration, mitochondrial content, metabolic profiles and tissue pathology. In the heart, FA increased concentrations of the TCA metabolite citrate (+ 4.2-fold, p < 0.01) and lowered ATP levels (- 1.9-fold, p < 0.001), confirming the inhibition of the TCA cycle by FA. High-resolution respirometry of cardiac mitochondria further revealed an impairment of mitochondrial complex V (CV)-linked metabolism, as evident by a reduced phosphorylation system control ratio (- 41%, p < 0.05). The inhibition of CV-linked metabolism is a novel mechanism of FA cardiac toxicity, which has implications for drug development and which NV354 was unable to counteract at the given dose. In the brain, FA induced the accumulation of ß-hydroxybutyrate (+ 1.4-fold, p < 0.05) and the reduction of mitochondrial complex I (CI)-linked oxidative phosphorylation (OXPHOSCI) (- 20%, p < 0.01), the latter of which was successfully alleviated by NV354. This promising effect of NV354 warrants further investigations to determine its potential neuroprotective effects.

Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds.

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds.

Fexapotide triflutate vs oral pharmacotherapy as initial therapy for moderate-to-severe benign prostate hyperplasia patients: a cost-effectiveness analysis.

BACKGROUND: To assess the price range in which fexapotide triflutate (FT), a novel injectable, is cost-effective relative to current oral pharmacotherapy (5 α-reductase inhibitor, α-blocker, 5 α-reductase inhibitor and α-blocker combination therapy) as initial therapy followed by surgery for moderate-to-severe benign prostate hyperplasia patients with lower urinary tract symptoms (BPH-LUTS). METHODS: We developed a microsimulation decision-analytic model to track the progression of BPH-LUTS and associated costs and quality-adjusted life years in the target population. The cost-effectiveness analysis was performed from Medicare's perspective with a time horizon of 4 years using 2019 US dollars for all costs. The microsimulation model considered treatment patterns associated with nonadherence to oral medication and progression to surgery. Model parameters were estimated from large randomized controlled trials, literature and expert opinion. For each initial treatment option, simulations were performed with 1000 iterations, with 1000 patients per iteration. RESULTS: Three upfront oral pharmacotherapy options are close in cost-effectiveness, with combination therapy being the most cost-effective option. Relative to upfront oral pharmacotherapy options, FT slightly increases quality-adjusted life years (QALY) per patient (1.870 (95% CI, 1.868 to 1.872) vs. 1.957 (95% CI, 1.955 to 1.959) QALYs). Under the willingness-to-pay (WTP) threshold of $150,000 per QALY, at price per injection of $14,000, FT is about as cost-effective as upfront oral pharmacotherapy options with net monetary benefit (NMB) $279,168.54. Under the WTP threshold of $50,000 per QALY, at price per injection of $5,000, FT is about as cost-effective as upfront oral pharmacotherapy options with NMB $92,135.18. In an alternative 10-year time horizon scenario, FT price per injection at $11,000 and $4,500 makes FT as cost-effective as oral pharmacotherapies. One-way sensitivity analysis showed this result is most sensitive to upfront therapy prices, FT efficacy and initial IPSS. At price per injections of $5,000, $10,000 and $15,000, the probability that FT is either cost-effective or dominant compared to upfront oral pharmacotherapy options using a WTP threshold of $150,000 per QALY is 100%, 93% and 40%, respectively. CONCLUSIONS: Compared to upfront oral pharmacotherapy options, FT would be cost-effective at a price per injection below $14,000, assuming a WTP threshold of $150,000 per QALY.

In vitro assessment of the dermal penetration potential of sodium fluoroacetate using a formulated product.

This paper presents experimental data on the skin absorption of sodium fluoroacetate from a formulated product using an in vitro approach and human skin. Sodium fluoroacetate is a pesticide, typically applied in formulation (1080) for the control of unwanted vertebrate invasive species. It has been assigned a Skin Notation by the ACGIH, and other international workplace health regulatory bodies, due to its predicted ability to permeate intact and abraded human skin. However, there is a distinct lack of experimental data on the skin absorption of sodium fluoroacetate to support this assignment. This study found that sodium fluoroacetate, as a formulated product, permeated the human epidermis when in direct contact for greater than 10 hr. A steady-state flux (Jss) of 1.31 ± 0.043 µg/cm2/hr and a lag time of 6.1 hr was calculated from cumulative skin permeation data. This study provides important empirical evidence in support of the assignment of a Skin Notation.

Photochemical C-F Activation Enables Defluorinative Alkylation of Trifluoroacetates and -Acetamides.

The installation of gem-difluoromethylene groups into organic structures remains a daunting synthetic challenge despite their attractive structural, physical, and biochemical properties. A very efficient retrosynthetic approach would be the functionalization of a single C-F bond from a trifluoromethyl group. Recent advances in this line of attack have enabled the C-F activation of trifluoromethylarenes, but limit the accessible motifs to only benzylic gem-difluorinated scaffolds. In contrast, the C-F activation of trifluoroacetates would enable their use as a bifunctional gem-difluoromethylene synthon. Herein, we report a photochemically mediated method for the defluorinative alkylation of a commodity feedstock: ethyl trifluoroacetate. A novel mechanistic approach was identified using our previously developed diaryl ketone HAT catalyst to enable the hydroalkylation of a diverse suite of alkenes. Furthermore, electrochemical studies revealed that more challenging radical precursors, namely trifluoroacetamides, could also be functionalized via synergistic Lewis acid/photochemical activation. Finally, this method enabled a concise synthetic approach to novel gem-difluoro analogs of FDA-approved pharmaceutical compounds.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

Comparison of 18F-FDG, 18F-Fluoroacetate, and 18F-FEPPA for Imaging Liver Fibrosis in a Bile Duct-Ligated Rat Model.

Developing sensitive diagnostic methods for a longitudinal evaluation of the status of liver fibrosis is a priority. This study is aimed at assessing the significance of longitudinal positron emission tomography (PET) imaging with 18F-labeling tracers for assessing liver fibrosis in a rat model with bile duct ligation (BDL). Twenty-one 6-week-old Sprague-Dawley male rats were used in this study. Longitudinal PET images using [18F]N-2-(2-fluoroethoxy)benzyl)-N-(4-phenoxypyridin-3-yl)acetamide ([18F]FEPPA) (n = 3), [18F]fluoroacetate ([18F]FAc) (n = 3), and 18F-fluoro-2-deoxy-D-glucose ([18F]FDG) (n = 3) were obtained at 0, 1, and 2 weeks after BDL. Biochemical assays, histological assays, immunohistochemical staining assays, and next generation sequencing analyses were also performed at 0 (n = 3), 1 (n = 3), 2 (n = 3), and 3 (n = 3) weeks after BDL, which demonstrated the severe damage in rat livers after BDL. Regarding [18F]FEPPA and [18F]FDG, there was a significantly higher uptake in the liver after BDL (both P < 0.05), which lasted until week 2. However, the uptake of [18F]FAc in the liver was not significantly different before and after BDL (P = 0.28). Collectively, both [18F]FEPPA and [18F]FDG can serve as sensitive probes for detecting the liver fibrosis. However, [18F]FAc is not recommended to diagnose liver fibrosis.

Evaluation of Class IIa Histone Deacetylases Expression and In Vivo Epigenetic Imaging in a Transgenic Mouse Model of Alzheimer's Disease.

Epigenetic regulation by histone deacetylase (HDAC) is associated with synaptic plasticity and memory formation, and its aberrant expression has been linked to cognitive disorders, including Alzheimer's disease (AD). This study aimed to investigate the role of class IIa HDAC expression in AD and monitor it in vivo using a novel radiotracer, 6-(tri-fluoroacetamido)-1-hexanoicanilide ([18F]TFAHA). A human neural cell culture model with familial AD (FAD) mutations was established and used for in vitro assays. Positron emission tomography (PET) imaging with [18F]TFAHA was performed in a 3xTg AD mouse model for in vivo evaluation. The results showed a significant increase in HDAC4 expression in response to amyloid-ß (Aß) deposition in the cell model. Moreover, treatment with an HDAC4 selective inhibitor significantly upregulated the expression of neuronal memory-/synaptic plasticity-related genes. In [18F]TFAHA-PET imaging, whole brain or regional uptake was significantly higher in 3xTg AD mice compared with WT mice at 8 and 11 months of age. Our study demonstrated a correlation between class IIa HDACs and Aßs, the therapeutic benefit of a selective inhibitor, and the potential of using [18F]TFAHA as an epigenetic radiotracer for AD, which might facilitate the development of AD-related neuroimaging approaches and therapies.

The Influence of Metabolic Inhibitors, Antibiotics, and Microgravity on Intact Cell MALDI-TOF Mass Spectra of the Cyanobacterium Synechococcus Sp. UPOC S4.

The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1-10 µM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10-5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

Development and Validation of a New Gas Chromatography-Tandem Mass Spectrometry Method for the Measurement of Enrichment of Glyoxylate Metabolism Analytes in Hyperoxaluria Patients Using a Stable Isotope Procedure.

Primary hyperoxalurias (PH) are inborn errors of glyoxylate metabolism characterized by an increase in endogenous oxalate production. Oxalate overproduction may cause calcium-oxalate crystal formation leading to kidney stones, nephrocalcinosis, and ultimately kidney failure. Twenty-four hour urine oxalate excretion is an inaccurate measure for endogenous oxalate production in PH patients and not applicable in those with kidney failure. Treatment efficacy cannot be assessed with this measure during clinical trials. We describe the development and validation of a gas chromatography-tandem mass spectrometry method to analyze the samples obtained following a stable isotope infusion protocol of 13C2-oxalate and 1-13C-glycolate in both healthy individuals and PH patients. Isotopic enrichments of plasma oxalate, glycolate, and glyoxylate were measured on a gas chromatography-triple quadrupole mass spectrometry system using ethylhydroxylamine and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) for analyte derivatization. Method precision was good for oxalate and glycolate (coefficients of variation [CV] were <6.3% and <4.2% for inter- and intraday precision, respectively) and acceptable for glyoxylate (CV <18.3% and <6.7% for inter- and intraday precision, respectively). The enrichment curves were linear over the specified range. Sensitivity was sufficient to accurately analyze enrichments. This new method allowed calculation of kinetic features of these metabolites, thus enabling a detailed analysis of the various pathways involved in glyoxylate metabolism. The method will further enhance the investigation of the metabolic PH derangements, provides a tool to accurately assess the therapeutic efficacy of new promising therapeutic interventions for PH, and could serve as a clinical tool to improve personalized therapeutic strategies.

Prospective evaluation of fexapotide triflutate injection treatment of Grade Group 1 prostate cancer: 4-year results.

PURPOSE: This study was undertaken to determine the safety and efficacy of fexapotide triflutate (FT) 2.5 mg and 15 mg for the treatment of Grade Group 1 prostate cancer. METHODS: Prospective randomized transrectal intraprostatic single injection FT 2.5 mg (n = 49), FT 15 mg (n = 48) and control active surveillance (AS) (n = 49) groups were compared in 146 patients at 28 U.S. sites, with elective AS crossover (n = 18) to FT after first follow-up biopsy at 45 days. Patients were followed for 5 years including biopsies (baseline, 45 days, and 18, 36, and 54 months thereafter), and urological evaluations with PSA every 6 months. Patients with Gleason grade increase or who elected surgical or radiotherapeutic intervention exited the study and were cumulatively included in the data analysis. Percentage of normal biopsies in baseline focus quadrant, tumor grades, and volumes; and outcomes including Gleason grade in entire prostate as well as treated prostate lobe, interventions associated with Gleason grade increase and total incidence of interventions were assessed. RESULTS: Significantly improved long-term clinical outcomes were found after 4-year follow-up, with percentages of patients progressing to interventions with and without Gleason grade increase significantly reduced by FT single treatment. Results in the FT 15-mg group were superior to the FT 2.5-mg dose group. There were no drug-related serious adverse events (SAEs). CONCLUSIONS: FT showed statistically significant long-term efficacy in the treatment of Grade Group 1 patients regarding clinical and pathological progression. FT 15 mg showed superior results to FT 2.5 mg. There were no drug-related SAEs; FT injection was well tolerated.

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography-triple quadrupole mass spectrometry method for determination of intact growth factor proteins.

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancer-related intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.

Astrocytic modulation of glutamatergic synaptic transmission is reduced in NTS of rats submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) activates chemoreceptors to produce cardiovascular and respiratory responses to bring the arterial partial pressure of O2 back to the physiological range. We evaluated the effect of SH (fraction of inspired O2 = 0.10, 24 h) on glutamatergic synaptic transmission and the interaction neuron-astrocyte in neurons of the nucleus tractus solitarii (NTS). Tractus solitarius (TS) fiber stimulation induced glutamatergic currents in neurons and astrocytes. SH increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) [-183 ± 122 pA (n = 10) vs. -353 ± 101 pA (n = 10)] and N-methyl-d-aspartate (NMDA) current amplitude [61 ± 10 pA (n = 7) vs. 102 ± 37 pA (n = 10)]. To investigate the effects of SH, we used fluoroacetate (FAC), an astrocytic inhibitor, which revealed an excitatory modulation on AMPA/kainate current and an inhibitory modulation of NMDA current in control rats. SH blunted the astrocytic modulation of AMPA [artificial cerebrospinal fluid (aCSF): -353 ± 101 pA vs. aCSF + FAC: -369 ± 76 pA (n = 10)] and NMDA currents [aCSF: 102 ± 37 pA vs. aCSF + FAC: 108 ± 32 pA (n = 10)]. SH increased AMPA current density [control: -6 ± 3.5 pA/pF (n = 6) vs. SH: -20 ± 12 pA/pF (n = 7)], suggesting changes in density, conductance, or affinity of AMPA receptors. SH produced no effect on astrocytic resting membrane potential, input resistance, and AMPA/kainate current. We conclude that SH decreased the neuron-astrocyte interaction at the NTS level, facilitating the glutamatergic transmission, which may contribute to the enhancement of cardiovascular and respiratory responses to baro- and chemoreflexes activation in SH rats. NEW & NOTEWORTHY Using an electrophysiological approach, we have shown that in nucleus tractus solitarii (NTS) from control rats, astrocytes modulate the AMPA and NMDA currents in NTS neurons, changing their excitability. Sustained hypoxia (SH) increased both glutamatergic currents in NTS neurons due to 1) a reduction in the astrocytic modulation and 2) an increase in the density of AMPA receptors. These new findings show the importance of neuron-astrocyte modulation in the excitatory synaptic transmission in NTS of control and SH rats.

amdS as a dominant recyclable marker in Cryptococcus neoformans.

While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors.

α-Peptoids as well as peptide/α-peptoid hybrids and peptide/ß-peptoid hybrids constitute major classes of proteolytically stable peptidomimetics that have been extensively investigated as mimetics of biologically active peptides. Representatives of lipidated peptide/ß-peptoid hybrids have been identified as promising immunomodulatory lead compounds, and hence access to these via protocols suitable for gram-scale synthesis is warranted to enable animal in vivo studies. Recent observations indicated that several byproducts appear in crude mixtures of relatively short benzyl-based peptide/ß-peptoid oligomers, and that these were most predominant when the ß-peptoid units displayed an α-chiral benzyl side chain. This prompted an investigation of their stability under acidic conditions. Simultaneous deprotection and cleavage of peptidomimetics containing either α-chiral α- or ß-peptoid residues required treatment with strong acid only for a short time to minimize the formation of partially debenzylated byproducts. The initial work on peptide/ß-peptoid oligomers with an alternating design established that it was beneficial to form the amide bond between the carboxyl group of the α-amino acid and the congested amino functionality of the ß-peptoid residue in solution. To further simplify oligomer assembly on solid phase, we now present a protocol for purification-free solid-phase synthesis of tetrameric building blocks. Next, syntheses of peptidomimetic ligands via manual solid-phase methodologies involving tetrameric building blocks were found to give more readily purified products as compared to those obtained with dimeric building blocks. Moreover, the tetrameric building blocks could be utilized in automated synthesis with microwave-assisted heating, albeit the purity of the crude products was not increased.

Controlling the Cleavage of Carbon-Carbon Bonds To Generate α,α-Difluorobenzyl Carbanions for the Construction of Difluoromethylbenzenes.

Controlling the cleavage of carbon-carbon bonds during a chemical reaction is a substantial challenge; however, synthetic methods that accomplish this objective produce valuable and often unexplored reactivity. We have designed a mild process to generate α,α-difluorobenzyl carbanions in the presence of potassium carbonate by exploiting the cleavage of C-C bonds during the release of trifluoroacetate. The initiating reagent is potassium carbonate, which represents an improvement over existing protocols that require a strong base. Fragmentation studies across substituted arenes and heteroarenes were conducted along with computational analyses to elucidate reactivity trends. Furthermore, the mildly generated α,α-difluorobenzyl carbanions from electron-deficient aromatics and heteroaromatic rings can react with aldehydes to create derivatives of difluoromethylbenzenes, which are valuable synthetic targets.

Evidence for the Formation of Difluoroacetic Acid in Chlorofluorocarbon-Contaminated Ground Water.

The concentrations of difluoroacetic acid (DFA) and trifluoroacetic acid (TFA) in rainwater and surface water from Berlin, Germany resembled those reported for similar urban areas, and the TFA/DFA ratio in rainwater of 10:1 was in accordance with the literature. In contrast, nearby ground water historically contaminated with 1,1,2-trichloro-1,2,2-trifluoroethane (R113) displayed a TFA/DFA ratio of 1:3. This observation is discussed versus the inventory of microbial degradation products present in this ground water along with the parent R113 itself. A microbial transformation of chlorotrifluoroethylene (R1113) to DFA so far has not been reported for environmental media, and is suggested based on well-established mammalian metabolic pathways.

Optimization of morphine extraction method for the assay of its urinary 3-glucuronideconjuguate by gas chromatography-mass spectrometry.

In the field of doping, a great interest is carried for the analysis of morphine, a powerful narcotic analgesic opiate which use is prohibited during competitions. In order to confirm the abnormal analytical result in our anti-doping laboratory, a sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was performed for the quantification of urinary morphine. As sample preparation is a key step for the determination of drugs in biological samples, the aim of this work consists of the optimization of the urinary human sample pretreatment conditions before quantification by GC/MS. Enzymatic hydrolysis associated with liquid-liquid extraction constitute the major pre-treatment steps. Our study has first focused on the optimization of the extraction solvents then to enzymatic hydrolysis which morphine is released from its glucuronide conjugated form. Onboard premiums, a study involving the effect of "amount of enzyme", "incubation temperature" and "duration of hydrolysis" was conducted. This univariate study has enabled us to evaluate the influence of each of these operating variables on the area ratio of morphine to the internal standard (Amorphine/AIS) response and to set the experimental fields for each one of them. Based on these results, an experimental design was established using the Box-Behnken model to determine, by multivariate analysis, the optimal operating conditions maximizing the "Amophine/AIS" response. After validation, the analysis of response surface makes it possible to set the optimum operating conditions, which the ratio "Amorphine/AIS" is maximized. The retained conditions for enzymatic hydrolysis are 160µl of Escherichia coli glucuronidase enzyme during 6hours of incubation at a temperature of 36°C. The solvent mixture Methyl-t-Butyl Ether/isopropanol (4:1, v/v) was selected since it has improved morphine extraction from the urinary matrix allowing a gain of 50% when compared to that used in our routine laboratory. Our developed extraction method can be successfully applied for our forensic anti-doping analysis of morphin in human sample urine.

Concentration-dependent changes to diffusion and chemical shift of internal standard molecules in aqueous and micellar solutions.

Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) is the most widely accepted internal standard for protein NMR studies in aqueous conditions. Since its introduction as a reference standard, however, concerns have been raised surrounding its propensity to interact with biological molecules through electrostatic and hydrophobic interactions. While DSS has been shown to interact with certain proteins, membrane protein studies by solution-state NMR require use of membrane mimetics such as detergent micelles and, to date, no study has explicitly examined the potential for interaction between membrane mimetics and DSS. Consistent with its amphipathic character, we show DSS to self-associate at elevated concentrations using pulsed field gradient-based diffusion NMR measurements. More critically, DSS diffusion is significantly attenuated in the presence of either like-charged sodium dodecyl sulfate or zwitterionic dodecylphosphocholine micelles, the two most commonly used detergent-based membrane mimetic systems used in solution-state NMR. Binding to oppositely charged dodecyltrimethylammonium bromide micelles is also highly favourable. DSS-micelle interactions are accompanied by a systematic, concentration- and binding propensity-dependent change in the chemical shift of the DSS reference signal by up to 60 ppb. The alternative reference compound 4,4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA) exhibits highly similar behaviour, with reversal of the relative magnitude of chemical shift perturbation and proportion bound in comparison to DSS. Both DSS and DSA, thus, interact with micelles, and self-assemble at high concentration. Chemical shift perturbation of and modulation of micellar properties by these molecules has clear implications for their use as reference standards.