This dissertation is mainly contributed with two main projects. The first project is based on aminomutase enzyme. Recent discovery of MIO-dependent aminomutases on the biosynthetic pathways of biologically active, medicinal compounds in plants and microorganisms raises interest in further understanding how they catalyze β-amino acid building blocks. A tyrosine aminomutase isolated from Japanese rice, Oryza sativa (OsTAM), converts α-tyrosine to β-tyrosine (75%) and makes an acrylate, p-coumarate (25%), as a by-product. OsTAM is the first TAM to have slight phenylalanine aminomutase (PAM) activity (3%). This may not be surprising since the active sites of OsTAM and TcPAM from Taxus plants differ by only two residues (Y125 and N446 of OsTAM compared to C107 and K427 of TcPAM, respectively) positioned similarly near the aryl ring of their substrates. We anticipated by changing key active site residues of OsTAM to nonpolar side chains found in TcPAM would improve the binding of substituted phenylalanine substrates. Another feature of MIO-aminomutases, highlighted in a previous study,1 is a hinge-gate inner loop that opens and closes the entry to the active site. We changed hydrophilic for more hydrophobic residues within the OsTAM loop like in TcPAM to make it function as a more efficient PAM and expected the mutants to produce a greater proportion of the β-amino acid over acrylate compared to that made by wild-type OsTAM. Our data suggested that a combination of active site mutants and loop mutants generally increased the turnover of OsTAM for para-substituted substrates over the other meta- and ortho- regioisomers to their corresponding cinnamates, and not to the β-amino acids, as the major products. These findings suggest that while active site residues may be involved primarily in creating broad substrate selectivity, their role along with that of the inner loop to parse the reaction toward β-amino acids remains elusive.The second project mainly focused on regioselective synthesis of ethyl dimethyl pyrazine. Alkylpyrazines are important heterocyclic compounds used as flavorants in the food and beverage industries. This study developed a regioselective synthesis of 2-ethyl-3,5-dimethylpyrazine (235-EDMP) over its 3-ethyl-2,5-dimethyl isomer (325-EDMP). Our first attempts explored how steric direct the coupling orientations between diamines to diketones to access 235-EDMP. Also, various physical parameters of the reaction conditions were changed, such as reduced temperature, the order-of-addition of reactants, and supplementation with chiral zeolites (Montmorillonite phyllosilicates) to template the orientation of the coupling partners to direct the regiochemistry of the reaction. Each reaction trial resulted in 50:50 mixtures of the ethyl dimethylpyrazine regioisomers. An alternative approach was explored to direct the regioselectivity of the reactions; acyloins (α-hydroxy ketone) replaced the diketone as the electrophilic coupling reactant used in the previous trial experiments. The hydroxy ketone reactants were made biocatalytically with pyruvate decarboxylase (E.C. 4.1.1.1). The coupling reaction between 2-hydroxypentan-3-one and propane-1,2-diamine resulted in the desired 235-EDMP at >70% (~77 mg total) relative to 30% 325-EDMP in the product mixture. The 3-hydroxypentan-2-one acyloin congener bio catalyzed and reacted with propane-1,2-diamine as proof of principle to make 325-EDMP (~60% relative abundance, ~57 mg) over the 235-EDMP. These results hinted toward a mechanism directed by the hydroxy ketone electrophilicity and the sterics at each nucleophilic center of the diamine.
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