Enantioselective homogeneous catalysts for the synthesis of fluorinated organic compounds

This thesis is divided into three main results chapters that reflect the path my research took. In the first results chapter, the first organocatalyst for the carbonyl-ene reaction was discovered and found to give high conversion using 1,3-bis(3,5-bis(trifluoromethyl)phenyl)thiourea. Various carbonyl and alkene precursors were examined in the ene reaction in both catalysed and uncatalysed reactions. It was found that ene reactions using fluoral and ethyl trifluoropyruvate give higher rates of reaction when compared to other carbonyl compounds. A novel enantiopure thiourea was synthesised and the ene reaction was catalysed enantioselectively to 33% e.e. In an attempt to catalyse the reaction to a further extent a new thiourea bonded to a P(=S)R2 group was developed. However, the intramolecular hydrogen bonding of this catalyst was thought to be so strong that this it did not catalyse the reaction. The synthesis of a chiral phosphoric acid was achieved but this was an unsuccessful catalyst in the ene reaction. Two component achiral thiourea and chiral acids were also examined in the ene and Mannich-type reaction. The new easily synthesised thiourea for this reaction has an interesting intermolecular hydrogen bonding coordination in the solid state. Asymmetric fluorination of ketoesters using palladium is a dynamic kinetic resolution. In the 2nd chapter cationic palladium complexes were synthesised and used to determine the optimum parameters for bidentate ligands in this reaction. Four carbon chain phosphines were found to give the highest conversion for this reaction among those ligands tested such as 1,4-bisdiphenylphosphinobutane (bite angle 99º). A new bis-phosphinous amide chiral ligand was developed with a bite angle of 96.7º. The dichloropalladium complex of this phosphine was isolated and structurally characterised. The use of the palladium complex in asymmetric fluorination was attempted however this was found to be unsuccessful. Mechanistic studies reveal that the formation of the desired cationic catalyst did not occur under conditions shown to work well for other palladium phosphine complexes. The ligand was investigated further in hydrogenation reactions. The phosphinous amide was protected as its borane and was used in the rhodium catalysed hydrogenation of alkenes to give high conversion and up to 93% e.e. The borane protected phosphinous amide was also found to catalyse the hydrogenation of acetophenone using copper complexes with up to 84% e.e for the hydrogenation of acetophenone, although conversion was quite low

Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings

Copper(II) binding by the earliest vertebrate gonadotropin‐releasing hormone, the type II isoform, suggests an ancient role for the metal

In vertebrate reproductive biology copper can influence peptide and protein function both in the pituitary and in the gonads. In the pituitary, copper binds to the key reproductive peptides gonadotropin‐releasing hormone I (GnRH‐I) and neurokinin B, to modify their structure and function, and in the male gonads, copper plays a role in testosterone production, sperm morphology and, thus, fertility. In addition to GnRH‐I, most vertebrates express a second isoform, GnRH‐II. GnRH‐II can promote testosterone release in some species and has other non‐reproductive roles. The primary sequence of GnRH‐II has remained largely invariant over millennia, and it is considered the ancestral GnRH peptide in vertebrates. In this work, we use a range of spectroscopic techniques to show that, like GnRH‐I, GnRH‐II can bind copper. Phylogenetic analysis shows that the proposed copper‐binding ligands are retained in GnRH‐II peptides from all vertebrates, suggesting that copper‐binding is an ancient feature of GnRH peptides

Project ATTAIN: Advancing Trauma-Informed Care for Youth with Intellectual and Developmental Disabilities and/or Gender Diverse Youth

Children with Intellectual and Developmental Disabilities (IDD) and/or gender diversity are at higher risk of experiencing trauma. Provider knowledge is lacking; trauma, disability, and LGBTQ+ resources are often siloed; and few providers screen for trauma in this population. This paper describes the design, delivery, and initial evaluation of Project ATTAIN (Access to Trauma-informed Treatment and Assessment for Neurodivergent and/or Gender-expansive Youth). ATTAIN is an ongoing 5-year state-wide initiative aiming to assess readiness to engage in new roles and practices over time; provide state-wide training and consultation in trauma, disability, and LGBTQ+-informed practices; install screening and assessment of trauma exposure and PTSD and quality of life into IDD and gender service settings; and include people with lived experience. A readiness assessment identified pre-training gaps between role responsibilities and practice engagement across five professional sectors serving our target population (n=39) in LGBTQ+-, disability-, and trauma-informed practices. We learned that specific sectors would benefit from introductory training to increase buy-in and promote role expansion; others would benefit from advanced instruction and implementation support. So far, we have trained 966 unique providers in trauma-informed care and have seen changes in the attitudes or perspectives of participants. Participants were highly satisfied with our provided training and saw increased knowledge across training. We screened 49 people in an IDD service setting for PTSD and quality of life. Two people with lived experience are active members of our research team, participating in project planning, training delivery, and manuscript authorship. Individuals who work with IDD and/or gender-diverse youth would benefit from increased training to expand their knowledge on LGBTQ+-, disability-, and trauma-informed practices. In year three, we intend to continue outreach and evidence-informed training focused on the intersection of trauma, IDD, and gender diversity. Ongoing evaluation of our outreach, training, and screening efforts will continue to inform program activities

Impact of tank background on the welfare of the African clawed frog, Xenopus laevis (Daudin)

The captive environment of a laboratory animal can profoundly influence its welfare and the scientific validity of research produced. The African clawed frog (Xenopus laevis) is a common model organism, however current husbandry guidelines lack supporting quantitative evidence. The visual environment is a fundamental aspect of a captive animal’s housing and may affect a number of physiological and behavioural responses. This is particularly important for species such as X. laevis where cryptic camouflage is a fundamental defence mechanism. Here male (n = 16) and female (n = 20) X. laevis were housed in tanks with ecologically relevant (black) and non-relevant (white) background colours and physiological and behavioural responses observed. Higher levels of water-borne corticosterone were observed in tanks with a white background compared to a black background in females (p = 0.047). Increased atypical active behaviours (Swimming: p = 0.042; Walling: p = 0.042) and a greater degree of body mass loss (p < 0.001) were also observed in the white background condition. Together these responses are indicative of increased stress of X. laevis when housed in tanks with a non-ecologically relevant background compared to an ecologically relevant background and suggest refined tank background colour may improve welfare in this species

Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells

T. brucei cathepsin-L increases arrhythmogenic sarcoplasmic reticulum-mediated calcium release in rat cardiomyocytes

Aims: African trypanosomiasis, caused by Trypanosoma brucei species, leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, the cardiac pathogenesis remains unknown. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter cardiac function independent of a systemic inflammatory/immune response. Methods and results: Using confocal imaging, T. brucei and T. brucei culture media (supernatant) caused an increased frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves) in isolated adult rat ventricular cardiomyocytes. Studies utilising inhibitors, recombinant protein and RNAi all demonstrated that this altered SR function was due to T. brucei cathepsin-L (TbCatL). Separate experiments revealed that TbCatL induced a 10–15% increase of SERCA activity but reduced SR Ca2+ content, suggesting a concomitant increased SR-mediated Ca2+ leak. This conclusion was supported by data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide, highlighting a role for CaMKII in the TbCatL action on SR function. Isolated Langendorff perfused whole heart experiments confirmed that supernatant caused an increased number of arrhythmic events. Conclusion: These data demonstrate for the first time that African trypanosomes alter cardiac function independent of a systemic immune response, via a mechanism involving extracellular cathepsin-L-mediated changes in SR function