Chemoenzymatic Synthesis of Chiral Amines by Carrier Protein-Dependent Enzymes

Abstract

Natural products have driven advancements in many fields for over a century, from methods for structural elucidation to the development of new synthetic reactions to replicate their complex scaffolds and biological activities. Because enzymes perform challenging synthetic transformations with superior chemo-, site- and stereoselectivity, scientists have more recently been inspired to leverage Nature’s biosynthetic machinery directly to make new compounds, establishing the emerging field of biocatalysis. This thesis describes studies with one broad class of proteins, carrier protein-dependent partner enzymes, to perform non-native reactions. Specifically, we developed biocatalytic platforms with two partner enzymes (PEs) SxtA AONS and AnaB to synthesize chiral amines chemoenzymatically. PEs, found in the biosynthetic pathways of polyketides and nonribosomal peptides, catalyze a variety of reactions to assemble diverse and elaborate natural products. However, they are not often employed to mediate non-native reactions because of the high cost of correct substrate activation. By understanding how carrier proteins (CPs) can be used efficiently in tandem with PEs or how PEs may operate in the absence of CPs, PEs can be incorporated into the syntheses of valuable small molecules. Chapter 1 summarizes multiple approaches toward utilizing PEs for synthetic purposes and strategies for employing non-native substrates. In Chapter 2, we characterized the native functions of the three PE domains within the polyketide-like synthase SxtA. We identified the correct starter unit and order of PE activity. Notably, the last domain, AONS transforms an amino acid into an alpha-amino ketone in a single step. We envisioned using this enzyme as a general tool to derivatize amino acids with and without the CP. We then optimized a CP-PE didomain platform with inexpensive acyl donors in Chapter 3 to perform the native reaction of SxtA AONS reaction scalably and to allow economical screening of the substrate scope. We observed ketone formation with multiple non-native thioesters and seven non-native amino acids but with very low overall conversion. We are improving the activity of SxtA AONS in a directed evolution campaign. Chapter 4 describes our investigations in alpha-deuteration of amino acids, a CP-free reaction. Deuterated amino acids are valuable precursors toward labeled pharmaceutical agents but challenging to synthesize. SxtA AONS installs a deuterium atom on the alpha-carbon of select alpha-amino acids and all alpha-amino methyl esters assayed. Preparative-scale reactions allowed for stereoselective chemoenzymatic synthesis of an isotopically labeled analog of the drug safinamide. Two SxtA domains, methyltransferase (MT) and AONS, were studied structurally and spectroscopically. In Chapter 5 we identify possible residues that lead to monomethylation in order to engineer additional dimethylation activity in SxtA MT. We also discuss UV-Vis studies with the AONS domain to understand the ketone-forming mechanism, elucidating the previously observed activity limitations and identifying possibilities for enzyme improvement. Finally, Chapter 6 details our work with AnaB, a PE that operates on proline, the only proteinogenic substrate incompatible with SxtA AONS. AnaB natively oxidizes CP-bound proline to an iminium ion that may be stereoselectively functionalized with an exogenous nucleophile in the preparation of chiral cyclic amines. We sequentially built a four-step catalytic cycle around AnaB to use the CP efficiently. The strategies presented in this thesis demonstrate the possibilities of leveraging CP-dependent PEs in chemoenzymatic synthesis. We anticipate that these approaches will also be applied to other PEs for the efficient, selective generation of chiral amines and other important synthetic building blocks.