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The aim of the thesis was to optimise the separation and purify an amino acid solution consisting of mono-valent amino acids. Amino acids have historically been separated by chromatographic separation for analytical requirements but separation commercially has not been possible due to the close physical and chemical properties of the amino acids. The study looks at two amino acids, isoleucine and alpha amino butyric acid and used operating and process variables to conclude whether a commercial application is possible. A 3m glass column operating at atmospheric conditions was set up in the development laboratory at SA Bioproducts for test work. Pulse tests were carried out to evaluate the separation of the individual amino acids and the samples were analysed using HPLC. The column incorporated a jacket which allowed for constant temperature during the experiments. Experiments started initially with the column being tested for hydraulic consistency and this was done using resin in the Na+ ionic form and passing a NaCI feed stream over the resin as a pulse. A base case was chosen and this using a feed volume of 0.1 bed volume, an eluant flow of 1 bed volume per hour, feed concentrations of 2% and an operating temperature of 75°C. The resin was used in the Na+ ionic form, with a particle size of 320llm, and a height of 1.5m. Results of the base case indicated a low resolution of 38%, and low recoveries at 11.85% at purities of 86%. Evident from the chromatogram was that isoleucine was held more strongly than AABA by the resin as isoleucine was eluted last. Increasing the concentration of the feed to 4% moved the peaks closer and caused more overlapping thereby decreasing the recovery. Decreasing the concentration resulted in better separation. From the results it was concluded that the separation was dependant on concentration. The next variable which was studied was the effects of changing the feed pulse volume. For this the feed volume was increased to 0.2BV and this ended up shifting the chromatogram to the right, with lower separation efficiency being measured. The effects of eluant flowrate were studied next. Decreasing the eluant flowrate to O.SBVIh resulted in sharper peaks and less overlapping. The conclusion reached was that the higher the contact time between the amino acids and the resin, the greater would be the separation efficiency. Increasing the flowrate of the eluant reduced the contact time and more overlapping was observed. The effects of temperature were also investigated. The experiment carried out at 90°C, showed very similar separation to the base case with higher recoveries. Decreasing the temperature to 30°C decreased the separation efficiency drastically. The experiments proved that an increase in temperature increased the kinetics, and allowed the amino acids to enter and exit the bead quicker. Once operating conditions have been evaluated, the resin was converted to the Ca2+ ionic form, and all other conditions were run according to the base case. ' From the chromatogram it was obvious that the amino acids were held more strongly by the resin and the retention time was increased thereby increasing the recovery. Changing the particle size of the resin from 320~m to 220~m increased the separation efficiency due to the faster kinetics. Increasing the height of the resin was attempted by adding more resin to the column. The separation efficiency was increased. Finally a optimised experiment was attempted where a 2% feed solution was used, feed volume was 0.1 BV, temperature of 90°C, eluate flow of O.SBV Ih, resin in the Ca2+ ionic form, with a particle size range of. 21 0-230~m, and resin height of 1.8m. The chromatogram indicated high peak separation efficiencies and high recoveries. The results indicated that the study was feasible. / Thesis (M.Sc.Eng.)-University of KwaZulu-Natal, Durban, 2005.

Amino acids are important building blocks for the synthesis of a large number of biologically active compounds and drugs. Amino acids with the pentacyclo-undecane (1) and trishomocubane (2) frameworks fall into the class of conformationally constrained non-natural amino acids. Conformationally constrained amino acids are found in many naturally occurring, biologically active compounds. It was found that incorporating cage structures into drugs induces a range of positive effects: promotes transport across the cell membrane, drugs can be designed to target the central nervous system, increased receptor site specificity, and retards metabolic degradation. In the light of this, it was decided to investigate the incorporation of cage amino acids into peptides. A synthetic route has been established for the efficient synthesis of amino acids 1 and 2, and for their incorporation into peptides. Several chiral macrocyclic crown ethers and related analogues have been shown to be capable of forming complexes enantioselectively with chiral organic ammonium salts. The design and synthesis of host chiral macrocycles which are able to distinguish between the enantiomers of guest organic ammonium salts is of interest in the areas that include synthesis of enzymes, electrodes for specific ions or molecules, drugs targeted for specific sites, and enantiomer separation. A synthetic procedure has been established for the synthesis of cage annulated chiral crown ethers derived from amino acids. The advantage of using cage compounds in crown ethers is due to increased rigidity, increased solubility in non-polar solvents and increased chirality. Various techniques for the determination of enantiomeric recognition have been studied and include NMR spectroscopy, fluorescence emission spectroscopy and computational methods. The cage crown ether 3 represents a typical example of these new cage annulated, chiral crown ethers. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

Cage compounds have attracted pharmaceutical and biological interest amongst others as anti-Parkinson agents. The serendipitous observation of the activity of 1-aminoadamantane 1 in Parkinsonian patients against selected viruses i.e. Herpes simplex Type I & II and Influenza A2-Asian viruses/Taiwan has increased the interest in cage compounds. This study involves the synthesis of the cage amino acid 14. Due to the insolubility of pentacyclo-[6.3.0.02,6.03,10.05,9]-undecane (trishomocubane) amino acid 14 in both polar and nonpolar solvents, including DMSO (d6), the synthesis of Fmoc-tris amino acid 50 was required for analysis. The Fmoc derivative of trishomocubane amino acid was also useful for controlled* coupling of the cage amino acid 14 to short peptides. The synthesis of the Fmoc-tris amino acid fluoride derivative is described as well as that of the tri-peptide (Ala-Ala-Ala). The incorporation of the Fmoc-tris amino acid fluoride in a tetra-peptide Ala-Ala-Ala-tris and in a hepta-peptide Ala-Ala-Ala-tris-Ala-Ala-Ala will also be presented. A computational chemistry project was undertaken using density functional theory (B3LYP) at the 6-31+G(d) level of theory, so as to enhance the understanding of the mechanism of esterification. Methanol, acetyl chloride and acetic acid were used in the model for simplicity. Four membered ring transition states were obtained with both acetyl chloride and acetic acid. A six membered ring transition state is facilitated by the selective use of one methanol molecule from the solvent. Both a concerted and step-wise mechanism are presented. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2006.

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. XLFL115D mutant completely eliminates ligation of all five substrates and previous X‑ray crystallography shows that XLF binds to XRCC4 via a “leucine lock” motif wherein L115 of XLF slips into a hydrophobic pocket in XRCC4. This makes the XRCC4-XLF interaction a good target to develop peptide inhibitors in order to radiosensitize breast tumor cells that are dependent on NHEJ to repair their DSBs after ionizing radiation exposure. Using mRNA display, we created a diverse library of 870 billion unique peptide sequences. After seven rounds of in vitro selection, the eluted fusions were cloned and sequenced. The results showed homology of sequences of five main families. We have selected representative peptides from those families (Pep 7.1-7.5), and several were chemically synthesized. However, none of these significantly inhibited XLF-dependent end joining in whole-cell extracts. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. The effectiveness of XLF-XLRCC4 inhibitors in blocking nonhomologous end joining remains to be determined.

Na maioria dos estudos de digestibilidade de aminoácidos em suínos usam-se animais de peso igual ou superior a 20 kg, isso se deve a menor dificuldade na implantação de cânulas íleo-cecais e a melhor recuperação pós-cirúrgica na fase do crescimento. No entanto, avaliar ingredientes com leitões mais jovens torna-se importante, visto que há limitações fisiológicas do trato gastrointestinal nesse período que podem afetar o seu desenvolvimento. Assim, 175 leitões desmamados, divididos em 7 ensaios experimentais com 25 animais cada, foram usados para determinar o balanço de nutrientes e da energia, a digestibilidade ileal aparente (AID) e estandardizada (SID) dos aminoácidos (AA) de sete ingredientes usualmente utilizados em dietas de leitões, com ou sem suplementação de multi-carboidrase (MC) e fitase (F). Os leitões foram desmamados aos 23 dias de idade e alojados em gaiolas para estudo de digestibilidade e metabolismo, permanecendo no experimento até os 45 dias de idade. Adaptação, coleta total de fezes e urina ocorreram do 10° ao 20° dia do período experimental e as amostragens do conteúdo ileal se deu ao abate, no 22° dia (45 dias de idade). Foi utilizado o delineamento experimental inteiramente casualisado com 4 tratamentos e cinco repetições. O leitão foi considerado como unidade experimental. As dietas experimentais consistiram de ingrediente teste sem enzimas e combinado a MC, F ou MC + F. Dois tipos de dieta referência foram usados como base para os cálculos do balanço nutricional e os coeficientes de digestibilidades aparente e estandardizada dos aminoácidos. Óxido de cromo (0,3%) foi usado como marcador indigestível nas avaliações dos aminoácidos. A enzima MC apresentava 10% de galactomananase, 10% de xilanase, 10% de β-glucanase, 60% de cevada maltada e 10% de α-galactosidase. A F era proveniente da fermentação de Saccharomyces cerevisiae com atividade de 10.000 FTU/g. Os ingredientes testados foram: farelo de arroz integral, soja integral micronizada, farelo de trigo, quirera de arroz, sorgo, milho e farinha de soja texturizada. Os resultados indicaram efeitos da combinação ingrediente e enzima exógena e as evidencias se deram de acordo com as características bromatológicas de cada um / Most of the studies, involving AA digestibility in pigs are used animals with 20 kg minimal weight or higher than. This is due difficulty to implant a simple T-cannula on distal ileum of younger pigs, besides a better post-surgical recovery in growing phase. However, ingredient evaluations with young pigs become important because there are physiological limitations in gastrointestinal tract that may affect its performance. Thus, 175 weaned pigs, divided into seven experimental trials with 25 animals each, were used to determine the nutrients and energy balance, the apparent (AID) and standardized (SID) ileal digestibility of amino acids (AA) from seven ingredients usually used in pig diets, with or without multi-carbohydrase (MC) and phytase (Phy) supplementation. Weaned piglets at 23 d old were housed in cages to studies digestibility and metabolism, remaining in the experiment until 45 d of age. Pig adaptation to feces and urine collection was 10 to 20 d experimental period and ileal content sampling at slaughter at 22 d (45 d old). A completely randomized experimental design was used with 4 treatments and 5 replicates. The pig was considered as an experimental unit. The experimental diets consisted of test ingredient as the sole source of protein with or without MC, Phy or MC+Phy. Two kind of reference diet were used to calculate the nutrient balance or AID and SID of AA. Titanium dioxide (0.3%) was used as indigestible marker. The MC enzyme had: galactomannanase, 10%; xylanase, 10%; beta-glucanase, 10%; malted barley, 60% and α-galactosidase, 10%. The Phy was obtained from the Saccharomyces cerevisiae fermentation with 10,000 FTU/g activity. The ingredients used were: rice bran, micronized full fat soybean meal, wheat bran, broken rice, sorghum, corn and texturized soybean meal. The results indicated effects from ingredients and enzymes combinations and the evidences occurred according to bromatological characteristics of each one

Thesis advisor: Eranthie Weerapana / Thesis advisor: Jianmin Gao / Proteins are composed of the 20 naturally occurring amino acids and are further modified by a variety of post-translational modifications (PTMS). Naturally occurring amino acids are diverse in structure and function. Catalytic amino acids, or nucleophilic amino acids, are of particular interest because of their contribution to chemical transformations in the cell. Synthetic covalent modification is a means to further functionalize or diversify proteins. These modifications, or enhancements, allow for improved understanding of protein structure, function and activity. For instance, isotope labeling of amino acid side chains in NMR studies enable investigators to study protein dynamics upon substrate or ligand binding. Fluorescence labeling is particularly useful to investigate protein cellular localization. Covalent modification is a useful tool to investigate the relative level of activity for protein known to be regulated by PTMs. An important feature of covalent modification reactions is site specificity, as this dictates the location, number of modifications, and protein targets. Tyrosine is of particular interest because it is both nucleophilic and aromatic. These characteristics contribute to the existence of tyrosine residues in both the protein surface and hydrophobic cores. Tyrosine is incorporated into proteins at a relatively low frequency. Unlike lysine, which is ubiquitous on protein surfaces, the low number of potential sites for general tyrosine modifications makes it an attractive site for surface bioconjugation modifications. A low number of surface modifications is less likely to perturb native protein function. Bioconjugation reactions give access to functionalizing the surface of proteins with moieties such as fluorophores, PEG, peptides, or drugs. Tyrosine is an attractive target for modifications because it is found in the active sites of a variety of enzymes such as sialidases, glutathione-S transferases, corticosteroid 11-beta-dehydrogensase, DNA topoisomerase, and ferredoxin-NADP+ reductase. Provided here is a survey of the known non-selective and selective synthetic chemical modification reactions for tyrosine. To investigate nucleophilic amino acids, Activity Based Protein Profiling (ABPP) may be implemented to investigate the role of these residues. ABPP utilizes small molecule covalent probes as a tool to selectively target enzymes in their active state. To investigate a protein of interest (POI) (or class of proteins) by ABPP, it is necessary to use a small molecule covalent probe that selectively reacts with the POI over other proteins within the proteome. Due to this requirement, it is necessary to expand the current ABPP probe toolbox to increase the coverage of what proteins in the proteome may be studied. Inspired by findings in the literature, our lab sought to explore the utility of various aryl halides for implementation in ABPP probes to overcome this limitation. This study revealed dichlorotriazine as a biologically relevant and reactive electrophile. A focus was placed on a dichlorotriazine containing probe library (LAS1-LAS20). LAS17 was discovered to be a potent and selective inhibitor of human glutathione S-transferase pi (GSTP1). Further studies revealed GSTP1 as a novel therapeutic target for the treatment of triple negative breast cancer. Other studies revealed several members of the dichlorotriazine library were found to covalently modify purified recombinant human aldolase A (ALDOA) in the presence of a complex cellular background. Additionally, LAS9 was identified as an inhibitor of ALDOA retro aldol condensation activity in vitro. Lastly, the final chapter highlights two collaborations in which tandem mass spectrometry experiments aid in the characterization of experimental data. In the first collaboration, a quantitative cysteine reactivity profiling method was used to characterize the selectivity of a cysteine reactive covalent NRF2-inducing small molecule, MIND4-17. In the second collaboration, analysis of tryptic mass spectrometry data enabled high resolution characterization of peptide sequencing for superfolder green fluorescent protein (sfGFP) expressed from observed internal nonsense suppression. Identification of the misincorporated amino acid facilitated the elucidation of the cross-talk mechanism. / Thesis (PhD) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Jianmin Gao / A common method of studying proteins is to introduce mutations into the amino acid sequence of the system. Incorporating phenylalanine analogs of varying degrees and sites of fluorination on the aromatic system gave substantial insight into the structure—function relationship of model peptide systems. By strategically placing tetrafluorinated phenylalanine mutants into the villin headpiece, HP35, increased thermodynamic and thermal stability was achieved. Using these highly but not fully fluorinated novel amino acid analogs allowed for the retention of the important ArH•••π interactions of the system. Furthermore, fluorinated amino acid residues were introduced into peptide systems known to form pores in lipid membrane systems. Certain fluorinated mutants of the membrane pore-forming peptides (MPP) showed increased membrane activity. Thus, fluorinated amino acids have tremendous potential to create hyperstable protein conformations, as well as increase the activity of proteins in membranes. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Chemistry.

Thesis advisor: Jianmin Gao / Thesis advisor: James P. Morken / This thesis includes two projects: "Novel perfluorinated aromatic amino acids: synthesis and applications" and "Thioflavin T dimers as novel amyloid ligands". I) Novel perfluorinated aromatic amino acids: synthesis and applications. Fluorinated amino acids serve as powerful tools in protein chemistry. Using the commercially available Boc-protected pentafluorophenylalanine, we synthesized a series of para-substituted tetrafluorophenylalanines via the regioselective SNAr reaction. These novel unnatural amino acids display useful and unique properties that can be applied to biological systems, including distinct 19F NMR signatures, pH-dependent amphiphilicity, lipid-binding selectivities, and halogen bonding capabilities. II) Thioflavin T dimers as novel amyloid ligands. Fluorescent molecules that specifically target amyloid structures are highly desirable for Alzheimer's disease research. We have designed a dimeric Thioflavin T that, through a reduced entropic penalty, has an improved binding affinity to Aβ amyloid by up to 70 fold. More importantly, the specificity and the "light-up" feature upon amyloid binding have not been sacrificed. Encouraged by the successful dimer design, we are further investigating the potential of amyloid-templated reactions to tailor-make ligands for amyloids. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

自由電子雷射器和傅裡葉變換質譜儀的組合使之成為一種研究生物分子和一些水和離子的溶解動力學的有力工具。而理論方法作為實驗的輔助手段常用于判斷一些分子的結構。在本論文中，我運用密度泛函理論和第一性分子動力學方法研究了陽離子化的氨基酸和一些水合陰離子的實驗紅外多光子吸收譜。 / 第二章研究了氫離子和鹼金屬離子化的天冬醯胺酸的紅外多光子吸收譜。以前有文獻用簡諧近似的方法研究過這個團簇，不過這種方法忽略了分子的動力學和非諧效應。我用第一性分子動力學方法重新研究了這個體系，結果顯示這種陽離子化的氨基酸的分子結構非常柔軟，在有限溫度下可以通過二面角的轉動和離子與氨基酸中的氧和氮原子距離的振動可以相互的轉化。而這些構型的相互轉化可以解釋實驗紅外吸收峰的展寬和相對強度變化趨勢。 / 第三章研究了水合碳酸氫根陰離子的紅外多光子吸收譜。以前的文獻指出，對於這種團簇，僅通過對比實驗和簡諧近似的紅外譜圖不能夠判斷是否只有一種異構體存在。應用第一性分子動力學方法，我發現簡諧近似紅外譜和實驗譜的不吻合之處，特別是水分子個數小於等於5時，水分子中氫原子的左右搖擺振動和來回搖擺振動吸收峰在實驗譜中的缺失。通過計算一系列異構體的熱能，我發現一些含量較少的異構體仍然對吸收峰有比較大的貢獻，考慮了這些結構的貢獻我們就可以理解為什麼隨著團簇尺寸的增加，一些吸收峰的強度有一定的變化趨勢。 / 最後兩章研究了水合磷酸二氫根的紅外多光子吸收譜。這是第一次我們和實驗組合作成果。在這個專案裡，我負責分析和全部的理論計算。對於這種團簇，實驗上測量了紅外譜的低頻和高頻區(n=1-4)。有趣的是，特別是體系只包含一個水分子時，兩個穩定的異構體均不能單獨解釋低頻和高頻的實驗結果。基於分子動力學的研究發現，有一個過渡態的吸收峰出現在了高頻區。其原因是氫原子的零點能和隧穿效應。與n=1類似,對於存在多個水分子的體系，實驗譜也揭示了低溫下的異構體效應，而這個效應使得簡諧效應完全失效。 / The combination of the FEL with FTICR makes it a powerful tool in researching the biochemical molecules and solvation dynamics of small hydrated ions. And theoretical methods are often employed in the determination of the molecular structures as auxiliary tools. In this thesis, density functional theory and ab initio molecular dynamics method have been applied to study the experimental infrared multiphoton dissociation (IRMPD) processes for cationized amino acids and hydrated anions. / The second Chapter is the AIMD simulated IR spectra for Asparagine cationized by H⁺, Li⁺, Na⁺, K⁺, and Cs⁺. The previous work simulated these type clusters with harmonic approximation in which the dynamics and anharmonicity effects were missed. My result shows that M⁺(Asn) complexes are very flexible and they could convert into each other at finite temperatures by torsional rotation and stretching the distances between the ion and N, O atoms in Asn. The broaden features and shift of the relative intensities of the vibrational profiles for these conformers are dependent on the structural flexibility of the complexes. / The third Chapter is the IRMPD spectra of the hydrated bicarbonate anion clusters for n=1-7. For some clusters, as mentions in the former harmonic results, comparing the experimental and calculated spectra does not always identify a single isomer as being responsible for the spectrum. By using AIMD technique, I found discrepancies between the harmonic and experimental spectra, especially for the absence of water wagging and rocking mode in the experimental result for n=1-5. By calculating the thermal energies of various isomers of each cluster, I found that the less dominate conformer also shows its large contributions to the vibrational profiles and this could explain the peak intensity trend as the cluster size increase. / The vibrational spectra for (HO)₂PO₂⁻(H₂O)n=1-4, 6, and 8 are presented in the last two chapters. It is the first time we collaborate with experiment group. In these works, I have done all the theoretical simulations. For these clusters, both the low and high (n=1-4) frequency regions of IRMPD spectra are measured. Interestingly for n=1, both the two stable isomer cannot alone represent both the low and high frequency profiles. Base on the AIMD simulations, I found that the existence of an unstable transient state shows it absorption profile in the high frequency region due to the zero point energy and tunneling effect of hydrogen atom. Similar to n=1, the IRMPD spectra of larger clusters (n>1) also show evidence for isomerization at cryogenic temperatures which leads to a complete breakdown of the harmonic approximation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Shoutian. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references. / Abstracts also in Chinese. / Sun, Shoutian.