Abstracts F1-F7

Category F.  Enabling Technologies

 

F1.  Reactivity Profiling: Sultams, Sulfonamides and Known Drugs as Electrophilic Probes

Collin D. Clay, Jung Ho Jun, and Paul R. Hanson*

Department of Chemistry, University of Kansas, Lawrence, KS, USA

Novel electrophilic probes have become a major interest to research the reactivity of nucleophilic residues in regulatory proteins. Specifically, the modification of cysteine as a target for Michael additions has been a pivotal focus as a treatment for Chagas disease and the modification of the ERK, Nrf2, and NF-kB biological pathways. Herein, we report the synthesis of fluorinated exo-vinyl sultams and sulfonamides for the analysis of covalent modification via thiols and cysteine Michael addition. Preliminary studies using 19F NMR as a tool to monitor nucleophile–electrophile reactivity patterns lead us to propose a platform for developing new electrophilic sulfur containing small molecules. Comparative studies utilizing 19F NMR analysis for the optimization of different thiol additions to previously synthesized five- and six-membered endo- and exo-vinyl sultams and known drugs as inhibitors targeted cancer therapy will also be discussed.

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F2.  A Divergent, Pot-economical and Library Amenable Approach for the Synthesis of C-, S-, and P-Containing α,β-Unsaturated 12–20-Membered Macrocycles

Gihan C. Dissanayake, Salim Javed, Dimuthu A. Vithanage, Ebony Onianwa, Arghya Ganguly and Paul R. Hanson*

Department of Chemistry, University of Kansas, Lawrence, KS, USA

A modular strategy for the synthesis of diverse macrocycles of various ring sizes (12–20-membered), substitution patterns and stereochemistry is reported. A modular tripodal coupling method has been developed to accomplish the synthesis of diverse electrophilic macrocycles. A 3-component coupling to unite three bi-functional fragments, namely a differentially-protected polyol subunit, an array of terminally differentiated P-stereogenic bicyclic phosphates, and olefinic α,β-unsaturated C-, S-, and P-based warheads. The modular approach includes several salient features, including (i) the use of differentially-protected polyol subunit to selectively generate various ring sizes through selective deprotection and coupling; (ii) chemoselective derivatization of the stereogenic carbinol centers allowing for FG and stereochemical attenuation; and (iii) late-stage variable warhead installation offering a wide range of attenuated C-, S-, and P-based electrophilic warheads.

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F3.  High Load Hybrid ROMP Reagents: Development and Applications in Facilitated Protocols

Saqib Faisal, Thomas A. Klein, Qin Zang, Jung H. Jun, and Paul R. Hanson*

Department of Chemistry, University of Kansas, Lawrence, KS, USA; The University of Kansas Center for Chemical Methodologies and Library Development (KU-CMLD), Shankel Structural Biology Center, University of Kansas, Lawrence, Lawrence, KS, USA

The development and applications of ROMP-derived hybrid oligomeric soluble/silica/magnetic phosphorus- and sulfur- based reagents in purification-free protocols is reported. The hybrid high-load Silica-ROMP oligomeric phosphate based alkylating reagents were successfully synthesized as free flowing solids for efficient alkylations of N-, O- and S-containing simple and complex scaffolds, including the generation of unique scaffolds in one-pot sequential protocols. In addition, developments of ROMP-derived oligomeric, reagents and scavengers immobilized on Co/C magnetic nanoparticles have been achieved via surface-initiated ROMP. Further development and utilization of new hybrid magnetic reagents are in progress for facilitated synthesis of small molecules, their applications in one-pot protocols, and applications in parallel synthesis and potentially automated technologies.

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F4.  Development of an On-line Microdialysis Microchip Electrophoresis-based Separation System for Monitoring Adenosine and its Metabolites

Shamal M. Gunawardhana and Susan M. Lunte

Ralph N. Adams Institute for Bioanalytical Chemistry, Department of Chemistry, University of Kansas, Lawrence, KS, USA

Traumatic brain injury (TBI) is the third highest cause of death in the USA. In addition, those who survive the primary impact of TBI can still suffer from severe secondary effects, such as cognitive disabilities and epileptic seizures. Unfortunately, the biochemical mechanism of the neurodegeneration causing these secondary effects is still not well understood. Purine compounds, including adenosine, inosine, and hypoxanthine, have been recognized as biomarkers for TBI. Monitoring the extracellular concentration changes of these biomarkers in the brain can be useful for better understanding of the biochemistry of neurodegeneration after a TBI. However, currently there is no clinical device available for the simultaneous monitoring of these biomarkers. In this study, we report the development of an on-line microdialysis (MD)-microchip electrophoresis (ME) separation-based sensor with amperometric detection (AD) for online and continuous monitoring of adenosine, inosine, and hypoxanthine, as well as guanosine, another purine compound found in brain extracellular fluid. Optimization of the ME separation conditions for these four biomolecules is described in addition to approaches that were utilized to lower the limits of detection (LOD).

The microchips used in this study were fabricated from polydimethylsiloxane (PDMS), and a carbon fiber (CF) working electrode was employed for the amperometric detection. Using a run buffer consisting of 35 mM boric acid at pH 10.0 with 1 mM sodium dodecyl sulfate and 15% dimethyl sulfoxide (v/v), we were able to successfully separate and detect all four biomarkers in less than 90 s with baseline resolution.

To achieve better separation efficiencies and a higher signal-to-noise ratio, both pseudo end-channel and off-channel detection were examined. In pseudo end-channel detection, the CF electrode was integrated into a PDMS platform placed at the end of the separation channel. However, this alignment resulted in significantly higher noise and background current than in off-channel detection. In off-channel detection, a polystyrene (PS) substrate with a Pd decoupler embedded before the CF working electrode was used to isolate the working electrode from the electric field. Using the hybrid PS/PDMS device, detection limits of 5, 10, 10, and 33 µM were achieved for hypoxanthine, adenosine, guanosine, and inosine, respectively.

To further improve sensitivity, CF electrodes modified with graphene oxide were investigated. The modified electrodes showed a significant signal enhancement for adenosine compared to unmodified electrodes with cyclic voltammetry. Currently, CF electrodes modified with electrochemically reduced graphene oxide are being tested with the ME system. Additionally, a dual-channel/dual-electrode microchip design is under investigation for lowering the LOD of the system by achieving on-chip background subtraction.

Ultimately, the developed ME-AD device will be coupled to microdialysis sampling for online monitoring of the four biomarkers in rat brain.

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F5.  Novel Triazole-Containing Tricyclic Sultams: Intramolecular C-Arylation of Triazoles

Viena Thomas, Maria Khan, Qin Zang, Elise Gao and Paul R. Hanson

Department of Chemistry, University of Kansas, Lawrence, KS, USA

An intramolecular C-arylation method for the synthesis of novel triazole-containing tricyclic sultams is reported. This diversity-oriented approach enables the synthesis of novel triazole sulfonamides and triazole-containing tricyclic sultams.  This de novo route involves an initial intermolecular Huisgen cycloaddition of an array of benzyl, alkyl and amino-alkly azides with various alkynyl sulfonamides, followed by an intramolecular C-arylation of the resulting triazole sulfonamides.  Furthermore, the synthesis of sulfonamides and sultams via functionalization of the aforementioned alkynyl sulfonamides utilizing diverging pathways such as double hetero-Michael addition and Diels-Alder reactions are also discussed.

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F6.  Development of an Electrochemically Generated Fluorescence Reporter System for Microchip Electrophoresis Based on a Bipolar Electrode

Manjula B. Wijesinghe1, Dulan B. Gunasekara2 and Susan M. Lunte1

1Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, KS, USA
2Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA.

Microchip electrophoresis coupled with amperometric detection (ME-EC) has been successfully employed for the determination of many important biomolecules, including antioxidants and reactive nitrogen and oxygen species. The limits of detection (LODs) for ME-EC are generally limited to the low micromolar range primarily due to the high noise generated by the separation voltage.  Therefore, the present LODs of ME-EC are not sufficient to quantify many biologically active analytes that are present at submicromolar concentrations. ME coupled with laser-induced fluorescence detection (LIF) has been shown to offer picomolar LODs due to the low background. However, LIF frequently requires the derivatization of the compound of interest with a fluorescent probe for detection, which is an extra step during the analysis and can lead to selectivity issues depending on the specificity of the probe. Bipolar electrodes, in which the electrochemical oxidation and reduction occur at the two extremities of the electrode without a direct ohmic contact, can be used to convert electrochemical current to an optical measurement such as fluorescence and electrogenerated chemiluminescence. The goal of this work is the development of an electrochemically generated fluorescence reporter system based on a bipolar electrode as a novel methodology that will provide lower LODs for electroactive compounds separated by ME.  

The system was first evaluated using a static detection system consisting of two reservoirs connected by a 15-μm Pt electrode, which was biased as a bipolar electrode. Ferrocene methanol was oxidized at the anodic pole while the non-fluorescent resazurin was reduced to fluorescent resorufin at the cathodic pole (a broadband light source was used for excitation). Under these conditions, the complete decay of the fluorescence signal took approximately 100 s. Therefore, it was concluded that a flow system is necessary to enhance the response time of the system and permit the detection of multiple analytes separated by electrophoresis. A 5 cm, 40 μm × 15 μm PDMS channel was used as the flow channel; it was connected to a vacuum pump at one end of the channel to pull the solution through the channel at a fixed flow rate. Additionally, a stronger excitation source (488 nm argon laser) was used to improve the signal intensity. The non-fluorescent molecule dichlorodihydrofluorescein diacetate was used as the reporter in the detector channel since it is electrochemically oxidizable to fluorescent dichlorodihydrofluorescein. Benzoquinone and resazurin were selected as model analytes for the ME separation. The separation and detection of benzoquinone (750 µM) and resazurin (25 µM) were successfully demonstrated using the newly developed system. The detection system will be further optimized and eventually applied to the detection of cellular immune response markers, such as peroxynitrite and nitrite, in single cells.

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F7.  Next Generation Sequencing at KU Genome Sequencing Core

Jennifer L. Hackett1,2,3,4, Melinda A. Branin1,2,3,4, Erik A. Lundquist1,2,4, Susan M. Lunte1,5,6

1Center for Molecular Analysis of Disease Pathways, 2Genome Sequencing Core, 3Higuchi Biosciences Center, 4Department of Molecular Biosciences, 5Department of Chemistry, 6Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS, USA

The Genome Sequencing Core (GSC) is one of three research core labs in the NIH COBRE Center for Molecular Analysis of Disease Pathways (CMADP) at the University of Kansas.  The major mission of the GSC is to provide researchers with access to next-generation sequencing (NGS) technologies. NGS, carried out in a massively parallel fashion, has been revolutionizing bio-medical research and used in a growing list of applications. Projects supported by the GSC include de novo genome assembly, genome re-sequencing for identification of mutations and polymorphisms, transcriptome analysis (RNA-seq), epigenomic and gene regulation studies such as ChIP-seq, Methyl-seq, small RNA discovery and analysis. The GSC enhances the genomics infrastructure already at KU by providing the Illumina MiSeq and higher throughput HiSeq 2500 sequencing capabilities to researchers at KU-Lawrence and across the region. The MiSeq system allows for more focused projects such as metagenomics and small genome sequencing. It has the capacity to generate 50 million reads of 300 bp per flow cell run (15 Gb data). The HiSeq 2500 system has the capacity to generate 3-6 billion reads of 100 bp per run on two eight-lane flow cells (600 Gb data). In its rapid mode, it can generate 1.2 billion reads of 250 bp per run on two two-lane flow cells (300 Gb data). To capture the full power of NGS, we provide a whole range of project support, from initial project consultation, sample quality check, library construction, cluster generation, sequence data generation, to preliminary data analysis.  For latest pricing, current job queue, or other info, visit the Core’s website: https://gsc.ku.edu/.

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