Enantioselective Hydroxylation of Benzylic C(sp; 3; )-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H; 2; O; 2; as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (; R; )-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor

Synthesis and binding studies of novel sigma receptor ligands

Abstract only availableSigma receptors are binding sites that are found in the brain, in the endocrine and immune systems, and also in the lungs, kidneys, intestines, muscles and especially the liver. They are classified into two subtypes, sigma1 and sigma2, both of which have unique characteristics. Sigma receptors in the central nervous system are thought to be involved in disorders such as psychoses, Alzheimer's disease, and schizophrenia. A number of human tumors also show high densities of sigma receptors. In this study, three novel compounds were synthesized with the intent of characterizing how their structural differences affect affinity for the sigma1 and sigma2 receptors. We investigated derivatives of a potent sigma1 selective agonist, 1-(3',4'-dimethoxyphenethyl)-4-(3''-phenyl propyl)piperazine, developed by Santen Pharmaceutical Co. Specifically, the 4'-methoxy moiety was replaced by benzyloxy, phenethyloxy and 3-phenylpropyloxy substituents. These were prepared by reaction of the corresponding 4'-phenol with base and treatment with phenethyl bromide, 3-phenylpropyl bromide or benzyl bromide. For the phenethyl and 3-phenylpropyl derivatives, a mixture of 40% KOH and tetrabutylammonium hydroxide (1M in MeOH) was used as the base. Column chromatography provided these target compounds in 81 - 94% purified yields. The benzyl derivative proved difficult to obtain using this procedure, and different conditions were used to synthesize this compound. The 4'-phenol was reacted with benzyl bromide and potassium carbonate in ethanol to give the benzyl ether in 35% yield after purification by column chromatography. All three compounds were characterized by 1H NMR, and were analyzed by elemental analysis and HPLC. Currently, competition receptor binding studies are being run on the synthesized compounds to measure their affinities for sigma1 and sigma2 receptors.NSF-REU/NIH Program in Radiochemistr

Recombinant Incretin-Secreting Microbe Improves Metabolic Dysfunction in High-Fat Diet Fed Rodents

peer-reviewedThe gut hormone glucagon-like peptide (GLP)-1 and its analogues represent a new generation of anti-diabetic drugs, which have also demonstrated propensity to modulate host lipid metabolism. Despite this, drugs of this nature are currently limited to intramuscular administration routes due to intestinal degradation. The aim of this study was to design a recombinant microbial delivery vector for a GLP-1 analogue and assess the efficacy of the therapeutic in improving host glucose, lipid and cholesterol metabolism in diet induced obese rodents. Diet-induced obese animals received either Lactobacillus paracasei NFBC 338 transformed to express a long-acting analogue of GLP-1 or the isogenic control microbe which solely harbored the pNZ44 plasmid. Short-term GLP-1 microbe intervention in rats reduced serum low-density lipoprotein cholesterol, triglycerides and triglyceride-rich lipoprotein cholesterol substantially. Conversely, extended GLP-1 microbe intervention improved glucose-dependent insulin secretion, glucose metabolism and cholesterol metabolism, compared to the high-fat control group. Interestingly, the microbe significantly attenuated the adiposity associated with the model and altered the serum lipidome, independently of GLP-1 secretion. These data indicate that recombinant incretin-secreting microbes may offer a novel and safe means of managing cholesterol metabolism and diet induced dyslipidaemia, as well as insulin sensitivity in metabolic dysfunction

Associations of sedentary behaviour, physical activity, blood pressure and anthropometric measures with cardiorespiratory fitness in children with cerebral palsy

Background - Children with cerebral palsy (CP) have poor cardiorespiratory fitness in comparison to their peers with typical development, which may be due to low levels of physical activity. Poor cardiorespiratory fitness may contribute to increased cardiometabolic risk. Purpose - The aim of this study was to determine the association between sedentary behaviour, physical activity and cardiorespiratory fitness in children with CP. An objective was to determine the association between cardiorespiratory fitness, anthropometric measures and blood pressure in children with CP. Methods- This study included 55 ambulatory children with CP [mean (SD) age 11.3 (0.2) yr, range 6-17 yr; Gross Motor Function Classification System (GMFCS) levels I and II]. Anthropometric measures (BMI, waist circumference and waist-height ratio) and blood pressure were taken. Cardiorespiratory fitness was measured using a 10 m shuttle run test. Children were classified as low, middle and high fitness according to level achieved on the test using reference curves. Physical activity was measured by accelerometry over 7 days. In addition to total activity, time in sedentary behaviour and light, moderate, vigorous, and sustained moderate-to-vigorous activity (≥10 min bouts) were calculated. Results - Multiple regression analyses revealed that vigorous activity (β = 0.339, p<0.01), sustained moderate-to-vigorous activity (β = 0.250, p<0.05) and total activity (β = 0.238, p<0.05) were associated with level achieved on the shuttle run test after adjustment for age, sex and GMFCS level. Children with high fitness spent more time in vigorous activity than children with middle fitness (p<0.05). Shuttle run test level was negatively associated with BMI (r2 = -0.451, p<0.01), waist circumference (r2 = -0.560, p<0.001), waist-height ratio (r2 = -0.560, p<0.001) and systolic blood pressure (r2 = -0.306, p<0.05) after adjustment for age, sex and GMFCS level. Conclusions - Participation in physical activity, particularly at a vigorous intensity, is associated with high cardiorespiratory fitness in children with CP. Low cardiorespiratory fitness is associated with increased cardiometabolic risk

The impact of El Niño events on the pelagic food chain in the northern California Current

The zooplankton of the northern California Current are typically characterized by an abundance of lipid-rich copepods that support rapid growth and survival of ecologically, commercially, and recreationally valued fish, birds, and mammals. Disruption of this food chain and reduced ecosystem productivity are often associated with climatic variability such as El Niño events. We examined the variability in timing, magnitude, and duration of positive temperature anomalies and changes in copepod species composition in the northern California Current in relation to 10 tropical El Niño events. Measurable impacts on mesozooplankton of the northern California Current were observed during seven of 10 of these events. The occurrence of anomalously warm water and the response of the copepod community was rapid (lag of zero to 2 months) following the initiation of canonical Eastern Pacific (EP) events, but delayed (lag of 2–8 months) following ‘Modoki’ Central Pacific (CP) events. The variable lags in the timing of a physical and biological response led to impacts in the northern California Current peaking in winter during EP events and in the spring during CP events. The magnitude and duration of the temperature and copepod anomalies were strongly and positively related to the magnitude and duration of El Niño events, but were also sensitive to the phase of the lower frequency Pacific Decadal Oscillation. When fisheries managers and biological oceanographers are faced with the prospect of a future El Niño event, prudent management and observation will require consideration of the background oceanographic conditions, the type of event, and both the magnitude and duration of the event when assessing the potential physical and biological impacts on the northern California Current.Keywords: climate change, northern California Current, food chain, trophic transfer, Eastern Pacific and Central Pacific El Niño, Pacific Decadal Oscillation, zooplankton, ENSO, copepo

Design and operation of a cryogenic charge-integrating preamplifier for the MuSun experiment

The central detector in the MuSun experiment is a pad-plane time projection ionization chamber that operates without gas amplification in deuterium at 31 K; it is used to measure the rate of the muon capture process $\mu^- + d \rightarrow n + n + \nu_\mu$. A new charge-sensitive preamplifier, operated at 140 K, has been developed for this detector. It achieved a resolution of 4.5 keV(D$_2$) or 120 $e^-$ RMS with zero detector capacitance at 1.1 $\mu$s integration time in laboratory tests. In the experimental environment, the electronic resolution is 10 keV(D$_2$) or 250 $e^-$ RMS at a 0.5 $\mu$s integration time. The excellent energy resolution of this amplifier has enabled discrimination between signals from muon-catalyzed fusion and muon capture on chemical impurities, which will precisely determine systematic corrections due to these processes. It is also expected to improve the muon tracking and determination of the stopping location.Comment: 18 pages + title page, 13 figures, to be submitted to JINST; minor corrections, added one reference, updated author lis

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution

Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macro molecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin (strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to &gt;8 g.L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i)With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C H bonds. Importantly, &gt;100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems