TOWARD THE DEVELOPMENT OF A BIOCATALYTIC PROCESS FOR THEPRODUCTION OF β-ARYL-β-AMINO ACIDSByNishanka Dilini RatnayakeWhile not abundant in nature, aromatic β-amino acids are found occasionally inpharmacologically important natural products such as andrimid, bestatin, and Taxol. In addition, they are used as important precursors for the synthesis of β-peptides and pharmaceuticals. Given the importance of β-amino acids, several methods are explored for the stereoselective synthesis of β-amino acids. Phenylalanine aminomutases, which isomerize (2S)-α-phenylalanine to β-phenylalanine with >99.9% ee, are efficient biocatalysts to produce enantiopure β-aryl-β-amino acids. The ultimate goal of this work was to use the knowledge from mechanistic investigations to rationally design Pantoea agglomerans phenylalanine aminomutase (PaPAM) as a biocatalystto produce industrially relevant compounds on scale.The 4-methylidene-1H-imidazol-5(4H)-one (MIO) cofactor is believed to serve as the electrophile in the isomerization reactions performed by phenylalanine aminomutases. Over several decades, debates have centered on whether π-electrons at the phenyl ring or the amino group of the substrate act as the nucleophile. The structure of PaPAM determined at 1.7 Ã
resolution revealed that the substrate (α-phenylalanine) and the product (β-phenylalanine) covalently bind to the MIO via the amino group. This data confirmed a PaPAM mechanism where the amino group of the substrate acts as the nucleophile.PaPAM was incubated with various isotopically labeled substrates to establish the stereochemistry and mechanism of the reaction. The reaction was found to proceed through removal, and interchange of the pro-(3S) hydrogen and the α-amino-group of the substrate, followed by intramolecular reattachment of the migration partners on the vicinal carbon from the same stereo-face that they originated from. Thus, PaPAM catalyzes its isomerization reaction with the inversion-of-configuration at both reaction termini.Variously ring-substituted α-phenylalanine analogues (19) were used to probe the substituent effects on the intermediate steps of PaPAM isomerization mechanism. Influence of the substituents on the catalytic rate (kcat) of PaPAM revealed concave-down correlations with Hammett substituent constants (σ). This trend suggested the rate-determining step changes from the step that eliminates hydrogen and amino group from the substrate to the 'hydroamination' step, based on the direction and magnitude of the electronic properties of the substituent. Theoretical calculations on analyzing enzyme-substrate interaction energies revealed that the steric effects in the protein-ligand adduct and within the ligand are dominant over electrostatic interactions when the substrate binds.The PaPAM E. coli whole-cell biocatalyst was shown to produce several non-natural (3S)-β-aryl-β-amino acids at >99.9% ee, with the highest turnover rate in M9 minimal medium at 16 C. The whole-cell biocatalyst biosynthesized 18 β-arylalanines with moderate to excellent converted yield (4-96%) at production levels of 8.5 - 235 mg·L-1 over 6 h, respectively. More notably, E. coli cells are reusable over at least five reaction cycles without a noticeable loss in activity and cell viability. This biocatalyst offers notable advantages over conventional synthetic methods and other biocatalysts because of its excellent enantioselectivity, broad substrate scope, single-step conversion, and sustainability.
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