Abstracts G1 - G7

Category G.  Enabling Technologies

 

G1.  A simple and efficient approach for mematinefluorescent derivative formation with potential applications to HPLC analysis

Leena Suntornsuk1,2,3, Pornpan Prapatpong1, ThanayuTecha-In1, Wantaporn Padungpuak1, Sawanya Buranaphalin1, Susan M. Lunte3

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University,
2Center of Excellence for Innovation in Drug Design and Discovery, Faculty of Pharmacy, Mahidol University,
3 Center for Molecular Analysis of Disease Pathways and Ralph N. Adams Institute of Bioanalytical Chemistry, University of Kansas

Memantine is an N-methyl-D-aspartate receptor antagonist recommended for treatment of moderate to severe Alzheimer’s disease.  Due to the lack of chromophore or fluorophore in memantine molecule, this work focuses on development of a novel procedure for memantine-fluorescent derivative formation enabling it to be monitored by a sensitive fluorescence detector.  4-(N-Chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulphonyl 2,1,3-benzoxadiazole (DBD-COCl) was chosen as a fluorescent probe since the carbonyl chloride of DBD-COCl could be easily react with the amine on memantine to form the fluorescent derivative.  Derivatization procedures were optimized and the proper condition was at 5:1 of DBD-COCl and memantine, at 60°C for 50 min.  The derivative structure was elucidated from mass spectrometry and infrared spectroscopy spectra confirming the amide bond formation of memantine-DBD-COCl.  The derivative was stable up to 24 h and could be monitored by high performance liquid chromatography (HPLC) coupled with a fluorescence detector using a VertiSepTM GES C18 column as the stationary phase, acetonitrile and water (80:20) as the mobile phase with a flow rate of 1 mL/min at room temperature.  Results indicate the successful formation of memantine-fluorescent derivative, which could be detected by fluorescence detection using the excitation and emission wavelengths at 430 and 520 nm, respectively.  The derivative was eluted at 4.50 min and was baseline separated from the DBD-COCl.  Preliminary validation data reveals good linearity (r2 0.99), repeatability (%RSD < 0.54) and accuracy (%R between 94 and 199%) with a limit of detection of 0.79 µg/mL (signal to noise ratio of 3).  Further improvement on sensitivity enhancement would benefit the analysis of memantine in biological samples.

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G2.  Biomolecular NMR Core Facility at University of Kansas

Asokan Anbanandam, Ph.D.

Biomolecular NMR Core Laboratory, Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF), University of Kansas, Lawrence KS

Biomolecular NMR (BNMR) Core Laboratory at KU was established in 2008 as part of the Center of Biomedical Research Excellence (COBRE) in Protein Structure and Function funded through NIH. We have two state of the art high resolution solution state NMR instruments, 600 and 800 MHz NMR.800 MHz NMR is equipped with cryo probe. Our 600 MHz NMR is equipped with 24 sample, sample automation unit and a broad band probe (BBO). BNMR core lab mission is to provide a full range of services in high-field NMR (solution state) including consultation in experiment design, data acquisition, processing, analysis and interpretation, for pilot studies, structural studies, dynamics studies, and molecular interaction studies, including fragment screening by STD-NMR and SPR (Surface Plasmon Resonance) for Fragment Based Drug Design (FBDD). We also provide training in instrument use, access to instruments for qualified users. All these services are available to investigators on a fee for service basis. Our capability to assist researchers is demonstrated through success stories featured in this poster.

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G3.  Determination of biogenic amines in fish using HPLC

Elton E. Melo Costa1,2, Victor H. O. Andrade 1, Jéssica J. P. Nascimento 1, Fabiane C. Abreu 1, Susan M. Lunte 2

1 Federal University of Alagoas – Institute of Chemistry and Biotechnology,
2The University of Kansas – Ralph N. Adams Institute for Bioanalytical Chemistry

Biogenic amines are substances formed through decarboxylation of specific amino acids by the action of microorganisms. Its presences in the food matrix, mainly in protein-rich foods such as fish, is directly correlated to the problems of intoxication and depend on the amount of these substances on food. A simple HPLC method was developed from the previously described method and modified for simultaneous determination of putrescine, cadaverine and histamine in frozen fish fillet. Amines were derivatized with OPA (pre column), and separated on a C-18 column using a fluorescence detector. The reference method used a gradient elution system with a mixture of methanol and phosphate buffer, the flow rate was 1.5 mL/min. The modified method changed the solvents to acetonitrile and water and the flow rate to 0.8 mL/min. The data confirmed that both methods obtained clear peaks for each investigated amine, however, the developed method presented a smaller total analysis time of 27.01 minutes while the total analysis time of the reference method was 62.01 minutes. Using method 2, calibration curves were obtained and the content of histamine, putrescine and cadaverine in fish sample were quantified.

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G4.  Development of High-Load, Hybrid Si-ROMP, Co/C Magnetic Reagents/Scavengers and Ligands.

Saqib Faisal a, Agnes Brandhofer b, Pradip K. Maity a, Diana S. Stoianova c, Oliver Reiser b, and Paul R. Hanson a*

aDepartment of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS, 66045. The University of Kansas Center for Chemical Methodologies and Library Development (KU-CMLD), 2034 Becker Drive, Shankel Structural Biology Center, West Campus, Lawrence, KS 66047.
bInstitute of Organic Chemistry, University of Regensburg, Universitätsstr. 31, 93053, Regensburg, Germany.
cMateria, Inc. 60 N. San Gabriel Blvd, Pasadena, CA 91107.

ROMP-derived oligomeric soluble/silica/magnetic phosphorus and sulfur based reagents for application in purification-free diversification protocols is reported. Hybrid Si-ROMP benzylic and heterocyclic phosphates and their corresponding derivatives were successfully synthesized as free flowing solids for efficient benzylation and triazolation. Building on the successful development of these ROMP-derived soluble and silica reagents/scavengers, we have further advanced them to supported magnetic Co/C nanoparticles utilizing surface-initiated ROM polymerization. Further developments of new hybrid magnetic reagents are in progress for their utility in synthetic transformations, facilitated synthesis of small molecules, applications in parallel synthesis and potentially automated technologies.

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G5.  Genome Sequencing Core Lab at KU-Lawrence

Xinkun Wang1,2,3, Jenny Hackett1,2, Erik Lundquist1,2,4, Susan Lunte 1,4,5

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

 

The Genome Sequencing Core (GSC) is one of three research core labs in the newly established NIH COBRE Center for Molecular Analysis of Disease Pathways (CMADP) at KU. The major mission of the GSC is to provide researchers with 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 Genome Sequencing Core enhances the genomics infrastructure already at KU, in the KU Genomics Facility and the Natural History Museum first generation sequencing facility, by bringing the astronomically high-throughput Illumina HiSeq 2500 sequencing capabilities to researchers at KU-Lawrence and across Kansas and the region. This next-generation sequencer has the capacity to generate 3-6 billion reads of 100bp per run of two eight-lane flow cells (600Gb data). In its rapid mode, it can generate 1.2 billion reads of 150 bp per run on two two-lane flow cells (120Gb data) in 27 hours. To capture the full power of NGS, we provide a whole range of project support, from project consultation, sample QC, library construction, cluster generation, data generation, to preliminary data analysis. For latest pricing, current job queue, or other info, visit the Core’s website: http://www.gsc.ku.edu/.

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G6.  Overview of Production of Protein Using Cell-free Systems

Anne Cooper, Philip Gao, Ph.D.

Center of Biomedical Research Excellence in Protein Structure and Function (COBRE-PSF), University of Kansas, Lawrence, Kansas
Del Shankel Structural Biology Center, 2034 Becker Drive, Room 1095, University of Kansas, Lawrence, KS 66047

One of the most important steps in protein research is production of the target protein. Cell based systems are mature tools that have long been used to express recombinant proteins by manipulation of the expression organisms. However, it is often challenging to find suitable cell systems that allow for rapid screening of conditions and constructs to produce properly folded, functional proteins in a cost effective manner. As a result, cell-free protein production emerged as an attractive alternative to cell-based protein expression methods because of its advantages including speed, simplicity, and adaptability to various formats. Efforts have been made in recent years to overcome a few major obstacles that had been preventing the system from being more widely used. These advances have led to the revitalization of cell-free expression systems to meet the increasing demands for protein production, and many research institutions and companies have developed unique and innovative cell-free systems. This workshop is designed for participants to learn the history and development of the cell-free method, and the updated techniques of various cell-free systems. Examples will be presented to demonstrate that the cell-free system can be a true alternative to cell based protein expression systems and offers a powerful technology for accelerating the production of recombinant protein.

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G7.  Surface Attachment of Genomic DNA for Downstream Forensic Applications

Tanya M Simms, Safa Alhussainalali, Anne Tacha, Matthew Antonik

Department of Physics & Astronomy, University of Kansas, Lawrence, Kansas

Current DNA profiling techniques rely on the accurate quantification of DNA present in biological evidence collected from a crime scene. Determining the amount of template DNA available for analysis is of utmost important given that each commercially available human identification kit specifies the amount of input DNA needed to produce optimum results. In most cases, approximately 1nanogram (1000 picograms) of template DNA is required for PCR amplification although some of the newer kits (e.g., GlobalFiler® and Powerplex® Fusion) can generate complete profiles using templates as low as 125 picograms. In this work, we use Total Internal Reflection (TIRF) microscopy, a single molecule technique that permits imaging of individual biomolecules (at concentrations of 10-100 picomolar) that are tethered to a glass surface. By using TIRF microscopy, we are able to assess both the quality (i.e., intact or broken) and quantity of the individual DNA molecules present in a sample. In addition, any contaminants or inhibitors, which are known to cause problems during the PCR amplification process, can be washed off following DNA surface attachment. To perform the proposed experiments, our first step was to determine a suitable method of attachment. Using lambda DNA, we assessed various attachment strategies, including both electrostatic (polylysine and divalent cations) and covalent (Amino-Amine and Thiol-Malelmide) methods, via PicoGreen, a free floating intercalator. Of these, the Thiol-Malemide combination was the most suitable, given that the images generated were clear with very little background fluorescence. As a result, this surface chemistry was then utilized for the final goal of the project, the attachment of genomic DNA to the surface. This part of the project was specifically undertaken because human genomic DNA, which is ~ 3 billion base pairs in length, forms a large clump when attached to the slide, making analysis of a specific region impossible. To overcome this obstacle, we fragmented the genomic DNA around the region of interest using a specific restriction enzyme. The goal is then to attach the genomic DNA to the slide via a hairpin structure, which we designed to consist of a 5’ Phosphate group, a genomic annealing region, and a thiol-modified linker region at the 3’ end. However, prior to the addition of the genomic DNA, we needed to confirm that the hairpin was anchored to the slide. This was done using a Cy5-labeled stretch of DNA that is complementary in sequence to the genomic annealing region. Presently, we are working on attaching and ligating the fragmented genomic DNA to the hairpin so that it can ultimately be used for downstream forensic applications.

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