The goal of the project is to utilize a well-studied small molecule-protein interaction to replace a C-H bond on the small molecule with a halogen atom (-X). The objective is to use the enzyme - Src protein kinase as a vice to hold a specific organic molecule in active site while subjecting the complex to C-H functionalization. The location(s) of the C-X functionalization will be compared to a control - the unbound small molecule, pyrrolopyrimidine (PPY) which is the strong Src inhibitor. The active site of the enzyme will be re-engineered to promote regio-selective C-X functionalization on a specific aromatic carbon atom. The goal is to exploit a small molecule-protein binding interaction as a template to effect electrophilic aromatic substitution (SEAr). The plan to achieve this is to (1) position a Lewis base moiety (a mutant methionine side-chain) in the Src active site to direct SEAr to a specific position on a bound PPY, and (2) combine in silico and directed evolution for the rapid discovery of Src mutants. The Src/PPY crystal structure is being assessed in silico for best candidate amino acid positions to mutate to methionine. The resulting library of mutant Src enzymes will be displayed on the surface of bacteria and incubated with fluorescent PPY. Successful binders will be isolated via Fluorescent Activated Cell Sorting. Thus far the gene for the Src kinase was obtained and cloned into Bacterial Surface Display (BSD) system recently developed in the Love Laboratory. PCR-based sub-cloning was verified by standard methods. Polyacrylamide gel electrophoresis was used to demonstrate protein expression. Favorable results from western blot analysis demonstrated that Src still functions as a protein kinase on the surface of E. Coli. Future plans are to incubate BSD Src with PPY and assess the C- H functionalization chemistry on bound and unbound PPY.

Using protein design to engineer a scaffold for regio-selective C-H functionalization

2019 Spring National Meeting

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