Palmer, David2022-12-1620222022-122022-12-16December 2https://hdl.handle.net/10388/14374Enzymes have the benefit of being able to catalyze reactions under mild conditions with a high degree of stereoselectivity and enantioselectivity that is sometimes difficult to achieve with traditional synthesis. NahE is an enzyme that is known to accept many aromatic and non-aromatic aldehydes and catalyze an aldol condensation reaction, but the full extent of the substrate scope was unknown. Here I report that benzaldehyde derivatives and even naphthaldehydes are accepted by NahE as substrates in the aldol condensation reaction with yields ranging from 30 - 100% but larger aldehydes were at the lower end of this range. This suggests that the size of the aldehyde is what limits activity. Furthermore, these enzymatic products can be decarboxylated with hydrogen peroxide to form cinnamic acid derivatives, which are useful products for the food/flavoring industry as well as substrates for other enzymes. These aldol condensation reactions could also be performed with the use of whole cells with little difference in yield and purity, which circumvents the needs for enzyme purification. With knowledge of the substrate scope, introduction of an amino substituent to the ortho-position of benzaldehyde was performed. This substrate was able to undergo an aldol reaction and subsequent transimination similar to another aldolase, dihydrodipicolinate synthase (DHDPS), to result in a heterocyclic product. These quinaldic acids were isolated with varying yields (12-92%) with many substrates isolated in >80% yield. Using HPLC, NMR and UV-Vis I was able to demonstrate that cyclization is occurring within the active site as I saw no evidence for release of the aldol product, which would lead to non-enzymatic cyclization. The fact that cyclization is occurring in the active site means that increased substrate steric bulk would result in lower yields and that is what is experimentally observed. Larger mono-substituted amino aldehydes show lower yields compared to smaller substituents at the same position and di-substituted amino aldehydes were poor substrates with yields <30%. After observing the remarkable substrate scope of NahE for many different types of aldehydes I wanted to use electrophiles other than aldehydes and settled on β-nitrostyrenes as they satisfy the aromatic preference of NahE in addition to being electrophilic. These electrophiles would be unable to form an elimination product and form chiral products. NahE was able to catalyze a nitro-Michael addition reaction with β-nitrostyrene and pyruvate with quantitative conversion to the 2-oxo acid product that could be isolated in ~85% yield, demonstrating that other electrophiles can be tolerated by this biocatalyst. Oxidative decarboxylation of the product to the corresponding carboxylic acid allowed near quantitative isolated yields over the same enzymatic reaction time (16 hours). The resulting product has a new stereogenic centre and characterization of the enantioselectivity of the reaction was performed. It was found that the enantiomeric ratio of the substituted product was 22:1 for the R-enantiomer as determined by HPLC on a chiral stationary phase as well as polarimetry. The enzymatic nitro-Michael reaction yields are more variable compared to the other reactions catalyzed by NahE with yields ranging from 15% to >99%. High enantioselectivity is observed when using para-substituted styrenes as substrates, and although enantioselectivity does erode for meta-substituted styrenes, this does highlight the utility of NahE for synthesis of enantioenriched products. Overall, this thesis highlights the use of a biocatalyst for the synthesis of α,β-unsaturated 2-keto acids from aldehydes, the synthesis of quinaldic acids from β-amino aldehydes, and the synthesis of chiral γ-nitro-β-aryl-2-keto acids from β-nitrostyrenes. These enzymatic products are formed via three distinctly different mechanisms and formed under mild, aqueous reaction conditions. The enzymatic products are also synthetically useful demonstrating the synthetic utility of biocatalysts.application/pdfenbiocatalysisNahEaldolaseasymmetric synthesisMichael additionThesis2022-12-16