The Analytical Chemistry team routinely purifies samples with material in the range of milligrams to grams. Major and minor components (< 0.1 percent) have been isolated for additional testing and characterization. The teams automated sample processing protocol allows for dispensing into 1D barcoded vials, Matrix 2D barcoded tubes and/or 96-well plates for efficient tracking, storing and testing. The entire process from receiving the sample to final plating is completed in less than one week. While the group uses a variety of liquid chromatographs to determine identity and purity, single quadrupole liquid chromatography/mass spectrometry instrumentation is utilized for high-throughput automated analysis. Due to the wide variety of analytes tested, the teams range of analytical detectors includes ultraviolet (UV), mass spectrometry (MS; positive and negative mode), and evaporative light scattering detector and fluorescence (ELSD). Time-of-flight mass spectrometry (TOF/MS) is employed to achieve formula confirmation and identity determination of unknowns. The chemical and enantiomeric purity of chiral compounds is routinely determined within the groups full scale chiral laboratory. Methods development with the chiral chromatography screening protocol involves utilizing seven different chiral stationary phases in conjunction with several mobile phase conditions facilitating the analysis and separation of nearly any chiral compound type. The use of an inline chiral detector allows for the determination of relative optical rotation. Furthermore, a vibrational circular dichroism (VCD) spectrometer has been acquired for the determination of absolute configuration. Matching the stationary phase of analytical columns with the corresponding stationary phase in both semi-preparative and preparative columns enables sample purification on a scale of up to hundreds of milligrams. The isolated enantiomers are returned as powders after chiral chromatography analysis to ensure purity of the material. As a means storing and organizing all analytical data, Analytical Chemistry developed Sample Management and Resource Tracking (SMART), which is a proprietary laboratory information management system (LIMS). SMART is a web-based management software utilizing a central server to store data from integrated instrumentation and automation equipment accessed through a unified web browser. While the initial focus was analytical data tracking and sample management, SMART has evolved into an all-encompassing system for sample submission, compound purification, compound processing, sample registration, compound inventory, sample tracking, data retrieval, and data management. As an expansion of SMART capabilities, the SMART Consumables, Acquisitions, and Reagent Tracker (SMARTCART) was created. SMARTCART is a web-based centralized system for the requisition, organization, and management of scientific related orders. By using this web-based program, scientists at DPI save significant time and effort related to obtaining the necessary materials related to their projects, which in turn saves money. The DPI Purchasing Liaisons are responsible for administrating these order requests and entering them into POTS. By utilizing SMARTCART, they will be able to do their job in a more efficient manner. SMARTCART is currently being used by the entire chemistry group at DPI (medicinal and analytical) and has begun being adopted by the DPI biology group. Fragment-based screening (FBS) is a complementary approach to high-throughput screening in the discovery of chemical entities for development into therapeutics. Fragment libraries are screened against protein targets associated with various disease states using established NMR techniques. In the development of its FBS program, DPI acquired two dedicated fragment libraries from Maybridge and CBC to augment the numerous fragments, which already exist as part of the NCATS library collection, as well as maximize structural diversity. Incorporation of these compounds into our compound management system was required for proper tracking and administration. This process included registration into the compound library, material transfer to barcoded storage vials, verification of sample quantities, and dissolution to create stock solutions. The standardization of these compounds was a highly labored endeavor involving the use of established automation technologies with liquid handling and lyophilization instrumentation. With the integration of the fragment libraries complete, the team has begun the massive task of compiling reference spectra for each fragment within the collection. In conjunction with the FBS program, Analytical Chemistry has started several collaborations, both internally and externally, which involves ligand-protein binding studies as a complement to the established medicinal chemistry programs. Over the past year, mass spectrometry capabilities at NCATS have significantly expanded with the acquisition and installation of two state-of-the-art mass spectrometry systems: an Agilent Chip-cube based nano-liter level separation QTOF MS system (6550) and an Agilent 1290-UPLC 6470 QQQ system. These systems allow Analytical Chemistry to meaningfully extend its scope of analysis into proteomics, metabolomics, and lipidomics, as well as intact protein and glycan analysis. Additionally, the QQQ system enables high sensitive quantitation analysis for small molecule samples (metabolites, lipids, compounds) and larger peptide/protein targets. When these mass spectrometers are coupled to the RapidFire high throughput sampling system, analysis capacity (qualitative and quantitative) within various biochemical and ADME assays could potentially increase to 5000-10,000 samples per day. These instrumentation have already been utilized for internal analytical chemistry projects and numerous collaborators within NCATS. Currently, Analytical Chemistry at DPI has access to thirteen mass spectrometry systems, which includes one GC-MS, eight HPLC-single Quad MS, one triple quad (QQQ) MS and three high resolution MS (two TOFs and one QTOF). Analytical Chemistry was a key contributor to the development and writing of a Chemical Biology Consortium (CBC) grant application to become a Dedicated Center, which supports the advancement of discovery projects within the NCI Experimental Therapeutics (NExT) program. NCATS was successful in its bid to become a CBC Dedicated Center and was awarded multi-year funding ($4M/yr; 2016-2020) for the fulfillment of this mission. Furthermore, this application has now become the source document for DPI facilities and capabilities.
| Liu, Li; Tang, Manshu; Walsh, Martin J et al. (2015) Structure activity relationships of human galactokinase inhibitors. Bioorg Med Chem Lett 25:721-7 |
