Developing deprotectase biocatalysts for synthesis.

Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include N-carbobenzyloxy (Cbz or Z) of amines and tert-butyloxycarbonyl (OtBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield. An overarching goal of biocatalysis is to use "green" or "enzymatic" methods to catalyse chemical transformations. One under-utilised approach is the use of "deprotectase" biocatalysts to selectively remove PGs from various organic substrates. The advantage of this methodology is the exquisite selectivity of the biocatalyst to only act on its target, leaving other FGs and PGs untouched. A number of deprotectase biocatalysts have been reported but they are not commonly used in mainstream synthetic routes. This study describes the construction of a cascade to deprotect doubly-protected amino acids. The well known Bacillus BS2 esterase was used to remove the OtBu PG from various amino acid substrates. The more obscure Sphingomonas Cbz-ase (amidohydrolase) was screened with a range of N-Cbz-modified amino acid substrates. We then combined both the BS2 and Cbz-ase together for a 1 pot, 2 step deprotection of the model substrate CBz-L-Phe OtBu to produce the free L-Phe. We also provide some insight into the residues involved in substrate recognition and catalysis using docked ligands in the crystal structure of BS2. Similarly, a structural model of the Cbz-ase identifies a potential di-metal binding site and reveals conserved active site residues. This new biocatalytic cascade should be further explored for its application in chemical synthesis.

Versatile Chemo-Biocatalytic Cascade Driven by a Thermophilic and Irreversible C-C Bond-Forming α-Oxoamine Synthase.

We report a chemo-biocatalytic cascade for the synthesis of substituted pyrroles, driven by the action of an irreversible, thermostable, pyridoxal 5'-phosphate (PLP)-dependent, C-C bond-forming biocatalyst (ThAOS). The ThAOS catalyzes the Claisen-like condensation between various amino acids and acyl-CoA substrates to generate a range of α-aminoketones. These products are reacted with ß-keto esters in an irreversible Knorr pyrrole reaction. The determination of the 1.6 Å resolution crystal structure of the PLP-bound form of ThAOS lays the foundation for future engineering and directed evolution. This report establishes the AOS family as useful and versatile C-C bond-forming biocatalysts.