The binding between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak is one of the key protein-protein interactions in the regulation of programmed cell death (apoptosis). Since it is well recognized that the BH3 domain of Bak adopts an amphipathic α-helix to interact with Bcl-xL through hydrophobic and electrostatic effects, conformational studies and possible applications of the α-aminoxy acid-containing peptides as mimics of the α-helix of Bak BH3 domain have been carried out. The main results are summarized below. Four short peptides ZT1?ZT4 containing alternating α-aminoxy acids/α-amino acids as the mimics of the α-helix of Bak protein were designed and synthesized. However, none of these four peptides, at the concentration of 25 μM, exhibited a significant inhibitory effect on the Bcl-xL inhibition test. Circular dichroism spectroscopic studies on ZT1?ZT4 as well as short model peptides N-minus, N-plus, C-minus and C-plus suggest that the proposed secondary structure, the 7/8 helix, is not stable in aqueous solutions. 1H NMR, 2D NMR and circular dichroism spectroscopic studies on the disulfide bond-constrained short peptides 4.7?4.9 with alternating α-aminoxy acids and α-amino acids suggest that a disulfide linker with three methylene units between adjacent α-amino acid residues could dramatically increase the stability of the 7/8 helix even in a mixed buffer/methanol solution. 1H NMR, 2D NMR and circular dichroism spectroscopic studies have also revealed that the hybrid soluble peptides C-free, N-free and Both-free containing α-amino acids and β-2,2-cyclopropyl-amino acids adopted a stable 8/8 helix in aqueous solution. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Eggshell of the African Ostrich, Struthio camelus, frequently occurs in African archaeological sites, some of which are beyond the limit of radiocarbon dating. In order to date those sites beyond the range of ¹⁴C dating using amino acid geochemistry, an understanding of the reaction kinetics governing amino acid chemistry must be achieved. The integrity of the eggshell matrix provides an excellent medium to investigate the kinetics of protein hydrolysis, amino acid decomposition, and isoleucine epimerization. Mathematical equations are derived from high temperature simulations of time and well-dated sites at ambient temperatures to determine rate constants for the reactions to a D/L ratio of 1.0. The reactions studied are Free and Total isoleucine epimerization, "classical" and "extended" hydrolysis, and the change in concentrations of free, bound, and total amino acids. Arrhenius plots aided in deriving the equations for each method which are then applied to predict kinetic parameters. Temperatures can be predicted within a 2°C range and time within 15% of its actual value if the appropriate method is used.

Described in this thesis is the isolation of two microorganisms containing a nitrile hydratase and amidase to effect the enantioselective hydrolysis of a-substituted nitriles to their corresponding amides and acids. Isolate NP3854 was identified as an atypical Rhodococcus sp. The nitrile hydratase proved to be non-selective for all the substrates tested. However, carboxylic acids with excellent enantiomeric excess were obtained from a large number of amides. X R~CN H nitr-il-e--h-y-d-r.a~tase ~X amidas.e RH CONH2 X R~"""CO H H 2 Optically active acids with an enantiomeric excess of, generally, >98 %, were obtained when X = NH2, Me and Cl, but proved to be racemic for OH and Br. R could be a variety of aromatic, cyclic and acyclic alkyl residues without adversely affecting the enantioselectivity. The pH-activity profile was determined for the amidase of NP3854 using propionamide as the substrate. From this data, coupled with inhibition studies, it may ,.tentatively be suggested that the amidase has a histidine residue in the active site, which may act as a general base for a serine amino acid. The pH-activity profile was determined for 2-amino-2-phenylacetamide 2b, and this suggested that the unprotonated form of the amine acted as the substrate. Within a pH range of 3 - 9 the enantiomeric excess remains high (>98 %) and experimentally invariant. The amidase was found to have a temperature optimum of 60°C and could tolerate 20 % THF with a loss of only 15 % activity. Attempts made to hydrolyse 4,5,6-amino nitriles and amides to the corresponding amino acids and isolate any reaction intermediates failed. This was presumed to be due to the large fraction of the unprotonated amine due to the higher pKa (- 9 - 10).

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