The objective of this thesis was to determine the molecular basis of neonatal multiple carboxylase deficiency (MCD) produced by an impairment in holocarboxylase synthetase (HCS) activity and the origin of the biotin-responsiveness that characterizes this disease. To determine HCS activity, I developed a peptide substrate and used the biotinylation system of E: coli to determine its properties. C-terminal fragments of the $ alpha$ subunit of human propionyl-CoA carboxylase (PCC-$ alpha$) were expressed in E. coli and site-directed mutagenesis was used to define the residues required for biotinylation by the bacterial biotin ligase, BirA. These experiments showed that the biotin region of PCC-$ alpha$ can act as an autonomous domain for biotinylation and suggested its use as substrate for human HCS. For the molecular characterization of MCD, I isolated several cDNA clones encoding human HCS by functional complementation of an E. coli mutant with a temperature-sensitive BirA. Comparison of the predicted amino acid sequence of HCS with bacterial biotin ligases allowed the identification of the putative biotin-binding domain of this protein. Mutation analysis of DNA from HCS deficient patients showed that most of the changes in the HCS sequence are clustered in the biotin-binding domain. All the patients tested in this study showed deficiency of HCS activity as determined using the PCC-$ alpha$ peptide as substrate for biotinylation. The biotin-responsiveness was demonstrated by obtaining a stimulation of HCS activity of MCD cells at high biotin concentrations while remaining unstimulated in extracts of normal cells. Together with the mutation studies, these results showed that neonatal MCD is caused by mutations in the biotin binding domain of HCS which reduce the affinity of the enzyme towards biotin. This change in the kinetic properties of HCS results in the inefficient biotinylation of carboxylases at physiological concentrations of biotin. The defect can be over

No description available.

This thesis examines the synthesis of α-fluoro-β-amino acids, and the influence of the constituent fluorine on the conformation and biological properties of β-peptide derivatives. Chapter One discusses the unique properties of the C-F bond, and applications of fluorine substitution in organic and medicinal chemistry. This is followed by a review of fluorinated analogues of α-amino acids, and how their incorporation into α-peptides has resulted in profound modifications, such as enhanced thermal and chemical stability, increased affinity for lipid bilayers, stronger self-association and improved biological activity. Experimental and theoretical data indicate two conformational effects associated with fluoroamides: the F-C-C(O)-N(H) moiety in α-fluoroamides adopts an antiperiplanar conformation, and in N-β-fluoroethylamides a gauche conformation between the vicinal C-F and C-N(CO) bonds is favoured. Chapter Two details the synthesis of a series of fluorinated β-peptides (2.13-2.24) designed to investigate the use of these stereoelectronic effects to control the conformation of β-peptide bonds. X-ray crystal structures were obtained for seven of these compounds and revealed the compounds had the expected conformations: when fluorine was positioned β to a nitrogen a gauche conformation was observed, and when fluorine was positioned α to a C=O group the structure adopted an antiperiplanar conformation. Thus, the strategic placement of fluorine can control the conformation of β-peptide bonds, and hence could be used to direct the secondary structures of β-peptides. The chapter is prefaced by an introduction to β-amino acids and the secondary structures of β-peptides. Chapter Three outlines the stereoselective synthesis of a series of α-fluorinated-β-amino acids. The synthesis of α-fluoro-β3-amino acids was achieved via direct fluorination of β3-amino acids with LDA and NFSI. The fluorination of N-Boc-protected β3-homophenylalanine, β3-homoleucine, β3-homovaline and β3-homoalanine all proceeded with good diastereomeric excesses (> 85 % de). However, the fluorination of N-Boc-protected β3-homophenylglycine occurred with a lower diastereomeric excess of 66%. Replacement of the Boc amine protecting group of β3-homophenylglycine with Cbz and Bz groups did not alter the stereoselectivity of the fluorination reaction, and substitution with an acetyl amine protecting group reduced the diastereomeric excess to 26%. The stereoselective synthesis of an α-fluoro-β2-homophenylalanine from 3-phenylpropanoic acid is also detailed. Conversion of the acid to the Evan's oxazolidinone followed by enantioselective fluorination and alkylation in high diastereomeric excess, and subsequent amination gave the α-fluorinated β2-amino acid. Chapter Four describes the enzyme assays carried out to assess the inhibitory activity of α-fluoro-β-amino acids, and the analogous non-fluorinated β-amino acids, against α-chymotrypsin. Both fluorinated and non-fluorinated β-amino acid derivatives were found to be competitive inhibitors of α-chymotrypsin, with Ki values in the low millimolar range. The fluorinated β2-homophenylalanine and β3-homophenylglycine derivatives (2.35, 3.26a, 3.43a and 3.44) were found to be more active against α-chymotrypsin than their non-fluorinated analogues (5.27, 3.24, 3.40 and 3.41), whereas the fluorinated β3-homophenylalanine methyl ester (2S,3S)-2.49 was inactive against α-chymotrypsin although the corresponding non-fluorinated derivative (S)-3.28 was a potent inhibitor. In Chapter Five a series of N-succinyl-β-amino acids-p-nitroanilides (5.8-5.13), containing both fluorinated and non-fluorinated β-amino acids, were designed and synthesised as possible substrates of α-chymotrypsin. β-Peptides are stable towards proteolytic hydrolysis, but the introduction of fluorine at the α-position in a β-amino acid was proposed to increase the activity of the adjacent amide bond, and thus make the β-peptide more susceptible to protease cleavage. However, the incorporation of fluorine had no influence on the proteolytic stability of compounds 5.8-5.13 as they were all found to be stable towards hydrolysis by α-chymotrypsin. Compounds 5.8, 5.9 and 5.13 were established as reversible competitive inhibitors of α-chymotrypsin Chapter Six is an experimental chapter and outlines the synthesis, purification and characterisation of the compounds prepared in this thesis.

This thesis is concerned with the use of kinetic resolution strategies for the preparation of enantiomerically pure materials. Chapter 1 introduces kinetic resolution. The limitations of conventional kinetic resolutions are described and the methods used to overcome these limitations are discussed. Chapter 2 presents a double kinetic resolution strategy where the recovered reactant from the first kinetic resolution is used as starting material in a second kinetic resolution. In the second kinetic resolution the major enantiomer present in the starting material is the faster reacting enantiomer. Application of this double kinetic resolution strategy to the Sharpless epoxidation is shown to enable enhanced product enantiomeric excesses to be obtained. Chapter 3 presents an alternative double kinetic resolution strategy where the product from the first kinetic resolution is used as starting material in a second kinetic resolution. In the second kinetic resolution the major enantiomer present in the starting material is the faster reacting enantiomer. Application of this double kinetic resolution strategy using lipase mediated esterification and hydrolysis reactions is shown to enable enhanced product yields to be obtained. Chapter 4 describes the preparation of an enantiomerically pure 2-substituted monoprotected propan-1,3-diol derivative via combination of an asymmetric synthesis and a kinetic resolution. Chapter 5 describes the preparation of the pheromone sulcatol in enantiomerically pure form via combination of an asymmetric synthesis and a kinetic resolution. Chapter 6 presents an investigation into the structure of lithium(α-methylbenzyl)- benzyl amide. Chapter 7 describes an attempted dynamic kinetic resolution of 2-substituted monoprotected propan-1,3-diol derivatives. Chapter 8 describes an attempted preparation of the iron crotonyl complex E-[(η⁵-C₅H₅)Fe(CO)(PPh₃)(COCH=CHCH₃)] in enantiomerically pure form via enzymic kinetic resolution.

Multiple carboxylase deficiency (MCD) results from a decreased activity of holocarboxylase synthetase (HCS) which is responsible for the biotinylation of the four biotin-dependent carboxylases found in humans. The disease can be treated with pharmacologic doses of oral biotin (biotin-responsiveness). The cDNA for HCS contains a biotin-binding domain deduced by analogy with the sequence and crystal structure of the E. coli BirA biotin ligase. E. coli birA$ sp-$ mutations causing biotin-auxotrophy all localize to this region. Of six point mutations I have identified in MCD patients, four localize to the biotin-binding region. In order to assess the HCS activity associated with patient mutations, I used an assay based on the expression of mutant HCS in E. coli. The method is based on the ability of mutant HCS to biotinylate the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase in a temperature-sensitive birA$ sp-$ E. coli strain using 3H-biotin as tracer. I have shown that all of the mutations cause a severe decrease in HCS activity. In addition, I have shown that five of the mutant HCS are biotin-responsive. These findings are a major contribution to the understanding of the mechanism of biotin-responsiveness.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of three mitochondrial and one cytosolic forms of biotin-dependent carboxylases in humans. Patients suffering from this autosomal recessive disease have Multiple Carboxylase Deficiency (MCD) with symptoms of life-threatening metabolic acidosis which, in almost all cases, can be successfully treated with pharmacologic doses of oral biotin. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS maybe targeted to the cytoplasm and mitochondria or that carboxylases are biotinylated in the cytoplasm prior to import into the mitochondria. In order to resolve the compartmentalization of HCS, 5' HCS cDNA sequences have been examined for a targeting signal and a candidate sequence was tested for its capacity to target mitochondria. Analysis of 5' cDNA reveals complex alternative splicing, none of which appear to contain mitochondrial targeting sequences. In addition, antibodies have been developed in order to perform immunochemical analysis of the subcellular distribution of HCS. Polyclonal antisera were raised against full length HCS as well as two peptides corresponding to a 20 amino acid region in the N-terminus and to the 20 amino acids preceding the stop codon. Immunohistochemical staining of human fibroblasts with the antibody to full length HCS gives cytosolic, mitochondrial and nuclear localization. Interestingly, analysis with the N-terminal antiserum reveals a large punctate staining pattern exclusively localized to the nucleus. The corresponding C-terminal antiserum reveals solid nuclear staining with some mitochondrial co-localization. Taken together, these results indicate the ubiquitous nature of HCS in human cells and also allude to a potential role for HCS in the nucleus of human cells.

No description available.

No description available.

Dietary intake data of 15 treated patients with phenylketonuria (PKU) (7-17 years) and six control siblings (6-14 years) were used to evaluate the effectiveness of medical foods to balance energy, protein, vitamin B-6, and individual amino acids from natural foods and to evaluate vitamin B-6 status. Four-day diet records were computer-analyzed and the contribution of medical foods and natural foods to the total diets of the patients was determined. A fasting blood sample and two 24-hour urine collections were obtained from each subject. In eight patients consuming a strict diet, natural foods provided 0.9 g protein and 39 mg phenylalanine (phe)/100 calories, a significant reduction from control intakes of 3.3 g protein and 153 mg phe/100 calories. However, plasma phe levels were above the acceptable treatment range. Medical foods were consumed in less than the recommended quantities to meet approximately 120% of the Recommended Dietary Allowances (RDAs) for protein. These foods contributed 73% of the total protein RDAs but only met 22% of the energy RDAs of patients consuming the strict diet. Taste qualities of certain L-amino acids (L-AAs; Lmethionine, L-glutamic and L-aspartic acids), which constitute the protein in elemental medical foods (EMFs) for school-aged patients, make acceptance of these products difficult. Intakes of each of these L-AAs were above nutritional standards and suggested that their levels could be safely lowered in EMFs. Natural foods and medical foods provided 0.057 and 0.046 mg vitamin B-6/g protein, respectively, above the 0.020 RDA standard and the mean control intake of 0.018 mg vitamin B-6/g protein. The mean plasma pyridoxal 5'- phosphate (PLP) concentration for the patients with PKU was over twice that of the mean control concentration and above literature values. However, more than half of the patients excreted less than 30% of their vitamin B-6 as 4-pyridoxic acid; values below the criterion suggested for inadequate status. This work provides data for a better understanding of vitamin B-6 metabolism and status in PKU and supports the design and testing of a new EMF to balance energy, protein, and amino acids from natural foods and which may improve dietary adherence. / Graduation date: 1991

Graduation date: 1984