Mechanistic Insights into the Palladium-Catalyzed Aziridination of Aliphatic Amines by C-H Activation.

Detailed kinetic studies and computational investigations have been performed to elucidate the mechanism of a palladium-catalyzed C-H activation aziridination. A theoretical rate law has been derived that matches with experimental observations and has led to an improvement in the reaction conditions. Acetic acid was found to be beneficial in controlling the formation of an off-cycle intermediate, allowing a decrease in catalyst loading and improved yields. Density functional theory (DFT) studies were performed to examine the selectivities observed in the reaction. Evidence for electronic-controlled regioselectivity for the cyclopalladation step was obtained by a distortion-interaction analysis, whereas the aziridination product was justified through dissociation of acetic acid from the palladium(IV) intermediate preceding the product-forming reductive elimination step. The understanding of this reaction mechanism under the synthesis conditions should provide valuable assistance in the comprehension and design of palladium-catalyzed reactions on similar systems.We are grateful to the EPSRC and Pfizer (A.P.S.) for a studentship and the ERC and EPSRC for fellowships (M.J.G.). We are also grateful to Dr Alex Thom and Dr David Pryde (Pfizer) for helpful discussions. The computational work was performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/), provided by Dell Inc. using Strategic Research Infrastructure Funding from the Higher Education Funding Council for England and funding from the Science and Technology Facilities Council. Mass spectrometry data were acquired at the EPSRC UK National Mass Spectrometry Facility at Swansea University.This is the accepted manuscript. The final version is available at http://pubs.acs.org/doi/abs/10.1021/jacs.5b05529

Towards micro-imaging with dissolution dynamic nuclear polarisation

Nuclear magnetic resonance (NMR) of small samples and nuclei with a low gyromagnetic ratio is intrinsically insensitive due to the received signal dependence on Boltzmann's statistics. This insensitivity can be partially overcome through the application of hyper polarisation techniques such as Dissolution Dynamic Nuclear Polarisation (D-DNP). It is hoped that the hyper polarised 13C signal received from labelled small molecules could facilitate imaging of metabolic and transporter processes in biological systems. In order to realise this, appropriate molecules and experimental hardware must be used. A detailed description of the experimental set-up used for carrying out DDNP is given and the system is characterised. the advantageous use of a dual iso-centre magnet system is elucidated with optimisation of acquisition of fast relaxing molecules. such a system allows for interrogation of processes with short relaxation times, not possible with traditional, stand-alone polarisers. To acquire the maximum amount of hyper-polarised 13C signal in an imaging experiment, parallel acquisition techniques have been implemented and the hardware designed with such goals in mind. Multiple coils have been used to allow accelerated image acquisition. As such this work has validated the SENSE algorithm for artefact free, image reconstruction on the micro-scale. These techniques require an array of coils which add to the complexity of the design of the probehead. Decoupling methods and array coil construction must be considered the methods used to ensure well isolated coils, such as geometric decoupling, are presented. The novel fabrication and implementation of micro-coils for imaging and spectroscopy of nL scale samples is presented this will help facilitate the acquisition of images showing metabolic processes in active transport in cells. By placing the coils close to the sample it is possible to gain sensitivity relative to the mass of the sample in question. To achieve signal detection on the order of nL a novel, exible micro-coil array has been fabricated and the results of NMR experiments carried out on both protons and 13C are shown. This is the final stage before integrating the coils with the D-DNP system. The acquisition of 13C signal with the micro-coils displays optimal electronic characteristics when compared with other detectors presented in the literature. The final goal of the work is to produce a system that is capable of micro imaging in small biological samples such as the Xenopus Oocyte with a view to monitoring metabolic processes and transportation without the need for the use of the large fluorescing proteins (GFP's) that have been used in previous work (1). The need for GFP's attached to metabolites results in the measured data being non-physical as the fluorescing protein is often much larger than the molecule being transported. It is hoped that the use of hyperpolarised small molecules (such as pyruvic acid) may be able to remove this need for GFP's in the study of metabolite transportation

Towards micro-imaging with dissolution dynamic nuclear polarisation

Nuclear magnetic resonance (NMR) of small samples and nuclei with a low gyromagnetic ratio is intrinsically insensitive due to the received signal dependence on Boltzmann's statistics. This insensitivity can be partially overcome through the application of hyper polarisation techniques such as Dissolution Dynamic Nuclear Polarisation (D-DNP). It is hoped that the hyper polarised 13C signal received from labelled small molecules could facilitate imaging of metabolic and transporter processes in biological systems. In order to realise this, appropriate molecules and experimental hardware must be used. A detailed description of the experimental set-up used for carrying out DDNP is given and the system is characterised. the advantageous use of a dual iso-centre magnet system is elucidated with optimisation of acquisition of fast relaxing molecules. such a system allows for interrogation of processes with short relaxation times, not possible with traditional, stand-alone polarisers. To acquire the maximum amount of hyper-polarised 13C signal in an imaging experiment, parallel acquisition techniques have been implemented and the hardware designed with such goals in mind. Multiple coils have been used to allow accelerated image acquisition. As such this work has validated the SENSE algorithm for artefact free, image reconstruction on the micro-scale. These techniques require an array of coils which add to the complexity of the design of the probehead. Decoupling methods and array coil construction must be considered the methods used to ensure well isolated coils, such as geometric decoupling, are presented. The novel fabrication and implementation of micro-coils for imaging and spectroscopy of nL scale samples is presented this will help facilitate the acquisition of images showing metabolic processes in active transport in cells. By placing the coils close to the sample it is possible to gain sensitivity relative to the mass of the sample in question. To achieve signal detection on the order of nL a novel, exible micro-coil array has been fabricated and the results of NMR experiments carried out on both protons and 13C are shown. This is the final stage before integrating the coils with the D-DNP system. The acquisition of 13C signal with the micro-coils displays optimal electronic characteristics when compared with other detectors presented in the literature. The final goal of the work is to produce a system that is capable of micro imaging in small biological samples such as the Xenopus Oocyte with a view to monitoring metabolic processes and transportation without the need for the use of the large fluorescing proteins (GFP's) that have been used in previous work (1). The need for GFP's attached to metabolites results in the measured data being non-physical as the fluorescing protein is often much larger than the molecule being transported. It is hoped that the use of hyperpolarised small molecules (such as pyruvic acid) may be able to remove this need for GFP's in the study of metabolite transportation

Labile Photo-Induced Free Radical in α-Ketoglutaric Acid: a Universal Endogenous Polarizing Agent for In Vivo Hyperpolarized 13 C Magnetic Resonance.

Hyperpolarized (HP) 13 C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only 13 C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive 13 C-molecules such as [1-13 C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the 13 C relaxation time in frozen HP 13 C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-13 C]lactate

Labile Photo-Induced Free Radical in α-Ketoglutaric Acid: a Universal Endogenous Polarizing Agent for In Vivo Hyperpolarized 13 C Magnetic Resonance.

Hyperpolarized (HP) 13 C magnetic resonance enables non-invasive probing of metabolism in vivo. To date, only 13 C-molecules hyperpolarized with persistent trityl radicals have been injected in humans. We show here that the free radical photo-induced in alpha-ketoglutaric acid (α-KG) can be used to hyperpolarize photo-inactive 13 C-molecules such as [1-13 C]lactate. α-KG is an endogenous molecule with an exceptionally high radical yield under photo-irradiation, up to 50 %, and its breakdown product, succinic acid, is also endogenous. This radical precursor therefore exhibits an excellent safety profile for translation to human studies. The labile nature of the radical means that no filtration is required prior to injection while also offering the opportunity to extend the 13 C relaxation time in frozen HP 13 C-molecules for storage and transport. The potential for in vivo metabolic studies is demonstrated in the rat liver following the injection of a physiological dose of HP [1-13 C]lactate

Photogenerated Radical in Phenylglyoxylic Acid for in Vivo Hyperpolarized ¹³C MR with Photosensitive Metabolic Substrates

WOS:000449239700056Whether for C-13 magnetic resonance studies in chemistry, biochemistry, or biomedicine, hyperpolarization methods based on dynamic nuclear polarization (DNP) have become ubiquitous. DNP requires a source of unpaired electrons, which are commonly added to the sample to be hyperpolarized in the form of stable free radicals. Once polarized, the presence of these radicals is unwanted. These radicals can be replaced by nonpersistent radicals created by the photoirradiation of pyruvic acid (PA), which are annihilated upon dissolution or thermalization in the solid state. However, since PA is readily metabolized by most cells, its presence may be undesirable for some metabolic studies. In addition, some C-13 substrates are photosensitive and therefore may degrade during the photogeneration of a PA radical, which requires ultraviolet (UV) light. We show here that the photoirradiation of phenylglyoxylic acid (PhGA) using visible light produces a nonpersistent radical t hat, in principle, can be used to hyperpolarize any molecule. We compare radical yields in samples containing PA and PhGA upon photoirradiation with broadband and narrowband UV-visible light sources. To demonstrate the suitability of PhGA as a radical precursor for DNP, we polarized the gluconeogenic probe C-13-dihydroxyacetone, which is UV-sensitive, using a commercial 3.35 T DNP polarizer and then injected this into a mouse and followed its metabolism in vivo

The effects of height and BMI on prostate cancer incidence and mortality:a Mendelian randomization study in 20,848 cases and 20,214 controls from the PRACTICAL consortium

Background\ud \ud Epidemiological studies suggest a potential role for obesity and determinants of adult stature in prostate cancer risk and mortality, but the relationships described in the literature are complex. To address uncertainty over the causal nature of previous observational findings, we investigated associations of height- and adiposity-related genetic variants with prostate cancer risk and mortality.\ud \ud Methods\ud \ud We conducted a case–control study based on 20,848 prostate cancers and 20,214 controls of European ancestry from 22 studies in the PRACTICAL consortium. We constructed genetic risk scores that summed each man’s number of height and BMI increasing alleles across multiple single nucleotide polymorphisms robustly associated with each phenotype from published genome-wide association studies.\ud \ud Results\ud \ud The genetic risk scores explained 6.31 and 1.46 % of the variability in height and BMI, respectively. There was only weak evidence that genetic variants previously associated with increased BMI were associated with a lower prostate cancer risk (odds ratio per standard deviation increase in BMI genetic score 0.98; 95 % CI 0.96, 1.00; p = 0.07). Genetic variants associated with increased height were not associated with prostate cancer incidence (OR 0.99; 95 % CI 0.97, 1.01; p = 0.23), but were associated with an increase (OR 1.13; 95 % CI 1.08, 1.20) in prostate cancer mortality among low-grade disease (p heterogeneity, low vs. high grade <0.001). Genetic variants associated with increased BMI were associated with an increase (OR 1.08; 95 % CI 1.03, 1.14) in all-cause mortality among men with low-grade disease (p heterogeneity = 0.03).\ud \ud Conclusions\ud \ud We found little evidence of a substantial effect of genetically elevated height or BMI on prostate cancer risk, suggesting that previously reported observational associations may reflect common environmental determinants of height or BMI and prostate cancer risk. Genetically elevated height and BMI were associated with increased mortality (prostate cancer-specific and all-cause, respectively) in men with low-grade disease, a potentially informative but novel finding that requires replication

Improved imputation of low-frequency and rare variants using the UK10K haplotype reference panel

Imputing genotypes from reference panels created by whole-genome sequencing (WGS) provides a cost-effective strategy for augmenting the single-nucleotide polymorphism (SNP) content of genome-wide arrays. The UK10K Cohorts project has generated a data set of 3,781 whole genomes sequenced at low depth (average 7x), aiming to exhaustively characterize genetic variation down to 0.1% minor allele frequency in the British population. Here we demonstrate the value of this resource for improving imputation accuracy at rare and low-frequency variants in both a UK and an Italian population. We show that large increases in imputation accuracy can be achieved by re-phasing WGS reference panels after initial genotype calling. We also present a method for combining WGS panels to improve variant coverage and downstream imputation accuracy, which we illustrate by integrating 7,562 WGS haplotypes from the UK10K project with 2,184 haplotypes from the 1000 Genomes Project. Finally, we introduce a novel approximation that maintains speed without sacrificing imputation accuracy for rare variants

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms