RAPID UPLC-ESI-MS/MS BASED ASSAY FOR DISCOVERY OF UDP-N-ACETYLMURAMOYL-L-ALANINE:D-GLUTAMATE (MurD) LIGASE INHIBITORSVjekoslava Car
, 2016, master's thesis
Abstract: A rapid, selective, robust and sensitive analytical assay method, operating in a short time frame with acceptable levels of precision, linear range and the accuracy necessary for successful Mur ligases inhibitors discovery, was developed.
An LC-MS/MS analytical procedure was designed for the determination of a MurD ligase reaction product (UMAG). The special focus of this work was on UDP-N-acetylmuramyl-L-alanine:D-glutamate ligase (MurD) activity. The assay method is especially valuable as an orthogonal (secondary) assay for the primary high throughput fluorescent-based assay screening of potential Mur ligase inhibitors. The LC-MS/MS assay is fully compatible with the components from the primary fluorescent-based assay and enables the analysis of the same samples by both methodologies. The presented LC-MS/MS assay procedure is used for the evaluation of the false positive hits (molecules) from the primary, fluorescence based, high throughput screening assay experiments. This is important for the elimination of false positive hits from the prohibitively expensive and time-consuming investigation process.
Method development describes the evaluation and optimization of the various stages of sample preparation, chromatographic separation, MS/MS determination and quantification. An enzyme reaction is performed in a 96-well plate. The quenched reaction mixture samples were spiked with an internal standard (phenacetin). The permeate was injected onto the U(H)PLC-MS/MS triple quadrupole system after sample ultrafiltration. Chromatographic separation was achieved on the ACQUITY UPLCTM HSS T3 column (100 x 2.1 mm i.d., 1.8 µm particle size) using an ammonium format buffer at pH 2.8 and acetonitrile as eluent. Elution initiated with an isocratic-hold for 1.1 min, followed by a two-step linear gradient of up to 3 min, giving a total run time of 5 min including equilibration. The flow rate was kept at a constant 0.4 mL/min.
UMAG quantitative analysis was performed by positive electrospray ionization, followed by tandem mass spectrometry (ESI-MS/MS). The analytical assay quantifies UMAG in a linear range from 0.25 to 20 µM using 70 µL of samples. Validation results demonstrated that UMAG concentrations can be accurately and precisely determined in samples from the primary assay.
Evaluation of inhibitory activities of compounds measured by both the fluorescence and the LC-MS/MS method demonstrated that the values were in a very good agreement. This analytical method can be used to screen a compound library at a defined concentration of each compound to obtain the percentage of inhibition, or with a series of compound concentrations to obtain inhibition potency of a compound (IC50). The selected Lek compounds no. 1 and 2 from the virtual screening campaign were presented, tested and further investigated due to the expression of significant MurD ligase inhibitory action acquired by primary high throughput tests.
This assay has been developed for MurD, but during development, chromatographic and MS/MS conditions for UM and UMA were studied and defined as well. Therefore, this analytical assay method can easily be applied to other Mur ligases (i.e. MurC, MurE) enzyme activity monitoring in the process of bacteria cell wall peptidoglycan formation. This method enables the identification of many different Mur ligase inhibitors in a continued search for new Gram positive and Gram negative bacteria antibiotics.
Keywords: Mur ligases, UDP-N-acetylmuramyl-L-alanine:D-glutamate (MurD) inhibitors, UNAM-Ala-Glu, LC-MS/MS, liquid chromatography, tandem mass spectrometry, antibiotics, drug discovery
Published: 23.09.2016; Views: 1602; Downloads: 111
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KINETICS OF CELLULOSE DEGRADATION STUDIED USING SIZE EXCLUSION CHROMATOGRAPHYAneta Balažic Fabjan
, 2016, master's thesis
Abstract: For more than five centuries, paper has been the predominant carrier of information and numerous medieval manuscripts bear witness of its durability. However, increasing demand for paper led to several changes in its production in the 19th century. High quality rag fibres were replaced by inferior wood-originating ones. Acid manufacturing technology was introduced which, due to its simplicity and low cost, continued to be used until the end of the 20th century. Inherently stable paper rapidly degrades in the presence of acids and its decay is further promoted by the poor storage conditions and environmental pollutants. As a result, the amount of degraded paper in libraries, archives and museums is reaching enormous proportions. In order to prolong the usable time of the vast quantities of original materials, paper collections may be deacidified and/or stored at lower temperatures. While preservation options are known, lack of the competent comparative studies leaves collection keepers hesitant of their use. The introductory part of the project is focused on development of analytical methodologies and model materials, representative of historical acid paper. As uniqueness and inherent value of cellulose-based cultural heritage limits the use of analytical methodologies to the non-destructive or micro-destructive ones. A new methodology for determination of the condition of paper was developed. The analytical technique-size exclusion chromatography for the first time allows us to reproducible determine the condition of paper which contains a significant amount of wood derived lignin. A few fibres suffice for the analysis, which renders the methodology suitable for characterisation of historical materials. The results of the research will represent the effect of deacidification processes with use of micro destructive analytical methodologies. As written word is all what we have for our legacy from generation to generation, evaluating preservation strategies for decaying collections, safekeeping and long term access to the endangered written cultural heritage is one of the most important facts.
Keywords: paper, size exclusion chromatography, kinetics, deacidification process
Published: 02.09.2016; Views: 1627; Downloads: 101
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Chemical and structural investigation of the cobalt phthalocyanineMatija Stupar
, 2015, master's thesis
Abstract: In the last two decades, studies on organic molecules mimicking substances of fundamental importance in nature, like chlorophyll or hemoglobin, have attracted researchers’ attention. These molecules are building blocks for a family of materials also referred to as “organic semiconductors”. Such compounds can be implemented in numerous applications, ranging from data-storage to light harvesting. Some of their fundamental advantages include low cost, light weight, relatively easy engineering and mechanical flexibility, compatible with bending plastic substrates.
In this thesis work we investigated the chemical, structural and electronic properties of cobalt phthalocyanines (CoPc). These molecules have promising applications in the field of magnetic data storage and spintronics in general, due to the ferromagnetic properties of the cobalt atom. Several techniques like photoemission core-level spectroscopy and valence band spectroscopy, together with X-ray absorption, have been used in order to determine the CoPc properties in gaseous phase, i.e. in the absence of interaction with the surrounding environment.
Another set of experiments was devoted to the commissioning of the CITIUS time-resolved photoemission setup, that will be used in future studies of CoPc molecules on surfaces.
Keywords: Cobalt phthalocyanine (CoPc), photoemission spectroscopy (PES), X-ray absorption spectroscopy (XAS), synchrotron radiation, laser, high order harmonic generation (HHG), time resolved spectroscopy
Published: 29.09.2015; Views: 2206; Downloads: 97
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The peroxy linkage in amorphous silicaBlaž Winkler
, 2014, master's thesis
Keywords: modeliranje, gostotna funkcijska teorija, GW približek, Bethe-Salpeterjeva enačba, silicijev dioksid, točkaste deformacije, peroksidna vez, magistrske naloge
Published: 22.01.2015; Views: 1727; Downloads: 91
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Epoxide ring opening of (3S,4R)-3-[(1R)-1-(tert- butyldimethylsilyloxy)ethyl]-4-[(1'r,2's,3'r)-1',2'-epoxycyclohex-3'-yl] azetidin-2-one with the nucleophiles under the microwave irradiationGregor Vilfan
, 2008, master's thesis
Keywords: ß-lactamase inhibitor, trinemi, stereoselektivna sinteza, regioselektivna sinteza, odpiranje epoksida, MAOS, magistrske naloge
Published: 15.10.2013; Views: 2010; Downloads: 86
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