Synthetic methods and biological applications of nitrogen heterocycles to compounds of biological interest.

Thalidomide analogues were synthesized utilizing optimized click conditions from 3-azidoglutarimide and an array of arylacetylenes or N-ethynyl/N-propargyl phthalimide derivatives. The intermediate, 3-azidoglutarimde, was pivotal and yielded a new and scalable synthesis. The reaction conditions utilized a copper sulfate/sodium ascorbate system in aqueous tetrahydrofuran to propagate the dipolar cycloaddition reactions between the azidoglutarimide and the alkynyl coupling partners. The first explored substrates were substituted arylalkynes to optimize the click reaction. Arylalkynes selected were to exhibit similar electron deficient rings to mimic phthalimide. Along with the synthesis of click thalidomide analogues, click analogue of the antiangiogenic and more potent teratogenic thalidomide analogue EM-12 was prepared. The isoindolinone group can act interchangeably with the N-phthaloyl nitrogen protecting group in amino acids. Amino acid intermediates using the N-isoindolinone group are used to make natural and unnatural a-amino acid derivatives using a two-carbon synthon. Selective benzylic oxidation was performed with the N-isoindolinone group being converted to the N-phthaloyl group (65–98%), which could be conveniently removed with hydrazine. For preparation of N-isoindolinone protected amino acids, an array of side chains was installed on the isoindolinone-protected glycine equivalent. This was accomplished through selective deprotonation to demonstrate the utility of the N-protected isoindolinone synthon (51–93%). Selective oxidation is employed on the benzylicN-isoinodolinone with OxoneÒ/KBr and successfully converted the N-isoindolinone group to the N-phthaloyl group in simple substrates, but substrates bearing unsaturated or electron-rich side chains respond poorly to the oxidation. The alkoxylation of chiral N-phenethyl hydroxyisoindolinones were synthesized with diethylaminosulfur trifluoride and an array of primary and secondary alcohols. The mechanism by which synthesis alkoxyisoindolinones are synthesized is through generation of an N-acyliminium ion to allow alkoxylation. The alkoxylated product exhibits a diastereomeric ratio influenced by the chiral N-phenethyl with a favored diastereomer. The stereochemistry and mixture of diastereomers were observed through this reaction

The development of self-determination: a qualitative exploration of the Indiana choice scholarship program

The 1960s launched the school movement of choice. Today, school choice is nationally threaded through many states serving a diversity of students. Indiana developed and implemented the Indiana Choice Scholarship Program (ICSP) policy in 2011 to align with these national initiatives to benefit students. The goal for Indiana was to ease the burden of private education costs for families regardless of a family’s income, but not all challenges within implementation have been remedied. The historical evolution of school choice currently focuses on the effectiveness of these state policies with regard to transitioning from the public to the private school for learners. Undergirded by Self-Determination Theory, this study focuses on high school students who matriculated from public to private ICSP Catholic schools. Participants were interviewed about their experience in both school settings after using the ICSP for at least one full school year. Participants reflected on their ability to make autonomous decisions, the importance of feeling connected to others, and their understanding of actions needed for career readiness. By implementing qualitative inquiry (Saldana, 2016), interviews explored perceptions about the ICSP, focusing on SDT. This approach aided in analyzing individual students’ perceptions behind the reasons why they chose to leave the public schools and enroll in Indiana’s Choice Scholarship Program. Qualitative inquiry allowed for a discussion about what had worked well, what had been a challenge, and what had helped ICSP students imagine college and career readiness after transferring schools. This design gave participants an opportunity to discuss choices regarding behaviors developing autonomy, relatedness to peers and mentors, and competence within academic programs and extracurriculars. Qualitative inquiry also discussed relevant topics that naturally evolved from conversations (Crotty, 2015) such as the COVID pandemic. The rationale for this selection was to explore how the ICSP had shaped a student’s opportunities for overall student success and college and career readiness using the SDT as a platform. The focus of this study was to have a discussion to understand if students see any benefit to a voucher scholarship program to attend an ICSP school. Cross-referencing responses added depth and understanding to the findings. Families still find school choice in Indiana difficult to sustain. Students in the study noted the challenge of transitioning from the public to the private school climates, commitment expectations, and financial costs associated with transferring to an ICSP school. While families still continue to struggle, Indiana continues to evolve the program to serve as many students K- 12 as possible. Regardless of personal tests, participants in this study were satisfied with their decisions to leave the public school to attend the ICSP school. They felt for their college and career success, the transition was necessary.Thesis (D. Ed.

A Comparison of Course Practices and Student Outcomes in Traditional Lecture Versus Modified Flipped Algebra I Classrooms

Abstract This study compared course teaching practices and student outcomes in 7 Algebra I classes taught in a modified flipped format and 7 classes in the same high school taught in a traditional lecture format during the same year. There were 4 teachers and about 200 hundred randomly assigned students in each format during the experimental year. The study also compared student outcomes of the students in the experimental year with those of students of the same teachers in the previous year when all classes were taught in the traditional lecture format. Based on interviews with the teachers, the study found that for the most part, the instructional flipped format was implemented with fidelity, i.e., the teachers carried out the modified flipped program as intended and in a manner that constituted a significant departure from the traditional lecture format. The study also found that students in the modified flipped classes scored higher on the Missouri State End of Course (EOC) algebra exams than those in the traditional lecture classes. However, examination of EOC data for the previous year revealed a similar pattern in the performance of each teacher’s students. In fact, the students of the teacher whose classes outperformed the others when taught using the modified flipped format outperformed the others by an even greater margin when all classes were taught using the traditional lecture format. Based on these findings, the study concluded that the observed difference in scores could not be attributed to the use of the modified flipped format

ADENYLATION AND TAILORING ACTIVITIES IN THE NONRIBOSOMAL PEPTIDE SYNTHESIS OF THE SIDEROPHORE PYOCHELIN

Pathogenic bacteria are becoming increasingly resistant to antibiotics. In response to this alarming trend, the scientific community must determine therapeutic targets. Essential nutrients, such as iron, are necessary for pathogens to survive and become virulent within a host system. One mechanism used by pathogenic bacteria to acquire iron from its surrounding environment is to produce low-molecular weight compounds which have a high affinity towards ferric iron. These compounds are called siderophores, and studies have shown their production to be essential for growth and virulence of some pathogens in iron-limited environments, such as the human host. Siderophores are often biosynthesized by nonribosomal peptide synthetases (NRPSs), which rarely have human homologs, making them attractive targets for novel therapeutics. NRPSs are enzymes utilized by bacteria, fungi, and plants to generate bioactive peptides. These bioactive peptides are not only used as secondary metabolites (toxins, pigments, siderophores) but have also found their way into the clinic as antibiotics, anticancer drugs, and immunosuppressants. To elicit their unique bioactivity, these peptides are tailored, making the compound chemically unique. Natural product chemists, metabolic engineers, and researchers in biochemistry and biotechnology work to exploit NRPS biosynthesis to generate new compounds for clinical use. This dissertation describes mechanistic and structural analyses of the adenylation and tailoring domains of the NRPS biosystem responsible for the production of pyochelin, a siderophore produced by antibiotic resistant Pseudomonas aeruginosa. A large portion of this work aims to better understand adenylation and “stuffed” tailoring didomains which lack structural characterization and have limited mechanistic understanding yet are ubiquitous in NRPS bioassembly. Pyochelin biosynthesis employs an adenylation-epimerase stuffed didomain in PchE and an adenylation-methyltransferase stuffed didomain in PchF. Substrate and product analogs were synthesized and steady-state adenylation, epimerase, and methyltransferase assays, along with onium chalcogen effects of the methyltransferase reaction, were used to characterize the adenylation-tailoring stuffed domains in pyochelin bioassembly. Similarly, substrate analogs were generated and used in steady-state kinetic and crystallography experiments with the stand-alone tailoring, NADPH-dependent reductase, PchG, and homolog, Irp3, of yersiniabactin biosynthesis. Finally, a steady-state adenylation assay was developed for the stand-alone salicylate adenylation enzyme, PchD, and potential warhead inhibitors were synthesized and co-crystallized laying the groundwork for future inhibitor design

Human Copper-Dependent Amine Oxidases

Copper amine oxidases (CAOs) are a class of enzymes that contain Cu2+ and a tyrosine-derived quinone cofactor, catalyze the conversion of a primary amine functional group to an aldehyde, and generate hydrogen peroxide and ammonia as byproducts. These enzymes can be classified into two non-homologous families: 2,4,5-trihydroxyphenylalanine quinone (TPQ)-dependent CAOs and the lysine tyrosylquinone (LTQ)-dependent lysyl oxidase (LOX) family of proteins. In this review, we will focus on recent developments in the field of research concerning human CAOs and the LOX family of proteins. The aberrant expression of these enzymes is linked to inflammation, fibrosis, tumor metastasis/invasion and other diseases. Consequently, there is a critical need to understand the functions of these proteins at the molecular level, so that strategies targeting these enzymes can be developed to combat human diseases

The FoxO Family in Cardiac Function and Dysfunction

The Forkhead family of transcription factors mediates many aspects of physiology, including stress response, metabolism, commitment to apoptosis, and development. The Forkhead box subfamily O (FoxO) proteins have garnered particular interest due to their involvement in the modulation of cardiovascular biology. In this review, we discuss the mechanisms of FoxO regulation and outcomes of FoxO signaling under normal and pathological cardiovascular contexts

Emerging evidence of coding mutations in the ubiquitin-proteasome system associated with cerebellar ataxias

Cerebellar ataxia (CA) is a disorder associated with impairments in balance, coordination, and gait caused by degeneration of the cerebellum. The mutations associated with CA affect functionally diverse genes; furthermore, the underlying genetic basis of a given CA is unknown in many patients. Exome sequencing has emerged as a cost-effective technology to discover novel genetic mutations, including autosomal recessive CA (ARCA). Five recent studies that describe how exome sequencing performed on a diverse pool of ARCA patients revealed 14 unique mutations in STUB1, a gene that encodes carboxy terminus of Hsp70-interacting protein (CHIP). CHIP mediates protein quality control through chaperone and ubiquitin ligase activities and is implicated in alleviating proteotoxicity in several neurodegenerative diseases. However, these recent studies linking STUB1 mutations to various forms of ataxia are the first indications that CHIP is directly involved in the progression of a human disease. Similar exome-sequencing studies have revealed novel mutations in ubiquitin-related proteins associated with CA and other neurological disorders. This review provides an overview of CA, describes the benefits and limitations of exome sequencing, outlines newly discovered STUB1 mutations, and theorizes on how CHIP and other ubiquitin-related proteins function to prevent neurological deterioration

Fuel-Stimulated Insulin Secretion Depends upon Mitochondria Activation and the Integration of Mitochondrial and Cytosolic Substrate Cycles

The pancreatic islet β-cell is uniquely specialized to couple its metabolism and rates of insulin secretion with the levels of circulating nutrient fuels, with the mitochondrial playing a central regulatory role in this process. In the β-cell, mitochondrial activation generates an integrated signal reflecting rates of oxidativephosphorylation, Kreb's cycle flux, and anaplerosis that ultimately determines the rate of insulin exocytosis. Mitochondrial activation can be regulated by proton leak and mediated by UCP2, and by alkalinization to utilize the pH gradient to drive substrate and ion transport. Converging lines of evidence support the hypothesis that substrate cycles driven by rates of Kreb's cycle flux and by anaplerosis play an integral role in coupling responsive changes in mitochondrial metabolism with insulin secretion. The components and mechanisms that account for the integrated signal of ATP production, substrate cycling, the regulation of cellular redox state, and the production of other secondary signaling intermediates are operative in both rodent and human islet β-cells

The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination

The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging

Rational inhibitor design for Pseudomonas aeruginosa salicylate adenylation enzyme PchD

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5′-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii