<div>The use of computational approaches to understand kinase substrate preference has been a powerful tool in the search to develop artificial peptide probes to monitor kinase activity, however, most of these efforts focus on a small portion of the human kinome. The use of high throughput techniques to identify known kinase substrates plays an important role in development of sensitive protein kinase activity assays.</div><div>The KINATEST-ID pipeline is an example of a computational tool that uses known kinase substrate sequence information to identify kinase substrate preference. This approach was used to design three artificial substrates for ABL, JAK2 and SRC family kinases. These biosensors were used to design ELISA and lanthanide-based assays to monitor in vitro kinase activity. The KINATEST-ID pipeline relies on a high number of reported kinase substrates to predict artificial substrate sequences, however, not all kinases have the sufficient number of known substrates to make an accurate prediction. </div><div>The adaptation of kinase assay linked with phosphoproteomics technique was used to increase the number of known FLT3 kinase variant substrate sequences. Subsequently, a set of data formatting tools were developed to curate the mass spectrometry data to become compatible with a command line version of the KINATEST-ID pipeline modules. This approach was used to design seven pan-FLT3 artificial substrate (FAStides) sequences. The pair of FAStides that were deemed the most sensitive toward FLT3 kinase phosphorylation were assayed in increasing concentrations of clinically relevant tyrosine kinase inhibitors. </div><div>To improve the automation of the mass spectrometry data analysis and formatting for use with the KINATEST-ID pipeline, a streamlined process was developed within a bioinformatic platform, GalaxyP. The data formatting tools used to process the FLT3 mass spectrometry data were converted into compatible versions to execute within the GalaxyP framework. This process was used to design four BTK artificial substrates (BAStide) to monitor kinase activity. Additionally, one of the BAStide sequences was designed in the lanthanide chelating motif to develop an antibody-free activity assay for BTK. </div><div>Lastly, a multicolored time resolved lanthanide assay was designed by labeling SYK artificial substrate and a SRC family artificial substrate to measure the activity of both kinases in the same kinase reaction. This highlighted the functionality of lanthanide-based time resolved assays for potential multiplexing assay development. </div><div><br></div>

<div> <div> <p>Continuous flow chemistry for organic synthesis is an emerging technique in academia and industry because of its exceptional heat and mass transfer ability and, in turn, higher productivity in smaller reactor volumes. Preparative electrospray (ES) is a technique that exploits reactions in charged microdroplets that seeks to accelerate chemical synthesis. In Chapter 2, the flow synthesis of atropine, a drug which is included in the WHO list of essential of medicines and currently in shortage according to the U.S Food and Drug Administration (FDA)is reported.The two steps of atropine synthesis were initially optimized separately and then continuously synthesized using two microfluidic chips under individually optimized condition.The telescoped continuous-flow microfluidics experiment gave a 55% conversion with an average of 34% yield in 8 min residence time. In Chapter 3, a robotic HTE technique to execute reactions in 96-well arrays was coupled with fast MS analysis. Palladium-catalyzed Suzuki-Miyaura (S-M) cross-coupling reactions were screened in this system and a heat map was generated to identify the best reaction condition for downstream scale up in continuous flow. <br></p><p><br></p><p>In Chapter 4, an inexpensive and rapid synthesis of an old anticancer drug, lomustine,was synthesized. Using only four inexpensive commercially available starting materials and a total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in two telescoped flow reactors. Sequential offline extraction and filtration resulted in 63% overall yield of pure lomustine at a production rate of 110 mg/h. The primary advantage of this approach lies in the rapid manufacture of lomustine with two telescoped steps to avoid isolation and purification of a labile intermediate, thereby decreasing the production cost significantly. A high throughput reaction screening approach based on desorption electrospray ionization mass spectrometry (DESI-MS) is described in Chapter 4 and 5 for finding the heat-map from a set of reaction conditions. DESI-MS is used to quickly explore a large number of reaction conditions and guide the efficient translation of optimized conditions to continuous flow synthesis that potentially accelerate the process of reaction optimization and discovery. Chapter 5 described HTE ofSNAr reactions using DESI-MS and bulk techniques with 1536 unique reaction conditions explored using both in DESI-MS and bulk reactors. The hotspots from the HTE screening effort were validated using a microfluidic system that confirmed the conditions as true positives or true.<br></p> </div> </div>