Site-Specific Protein Immobilization Using Unnatural Amino Acids

Protein immobilization confers the advantages of biological systems to a more chemical setting and has applications in catalysis, sensors, and materials development. While numerous immobilization techniques exist, it is optimal to develop a well-defined and chemically stable methodology to allow for full protein function. This paper describes the utilization of unnatural amino acid technologies to introduce bioorthogonal handles in a site-specific fashion for protein immobilization. To develop this approach a range of solid-supports, organic linkers, and protein immobilization sites have been investigated using a GFP reporter system. Overall, a sepharose resin derivatized with propargyl alcohol has afforded the highest yields of immobilized protein. Moreover, an unnatural amino acid residue protein context has been demonstrated, signifying a necessity to consider the protein site of immobilization. Finally, a resin-conferred stabilization was demonstrated in several organic solvents

Development of Solid-Supported Glaser–Hay Couplings

While the Glaser–Hay coupling of terminal alkynes is a useful reaction, several issues associated with chemoselectivity preclude its widespread application in synthetic chemistry. To address these issues, a solid-supported Glaser–Hay methodology was developed to afford only asymmetric diyne products. This methodology was then applied to a series of immobilized alkynes with a diverse set of soluble alkynes to generate an array of heterocoupled products in high yields and purities

Application of the Solid-Supported Glaser–Hay Reaction to Natural Product Synthesis

The Glaser–Hay coupling of terminal alkynes is a useful synthetic reaction for the preparation of polyynes; however, chemoselectivity issues have precluded its widespread utilization. Conducting the reaction on a solid-support provides a mechanism to alleviate the chemoselectivity issues and provide products in high purities and yields. Moreover, the polyyne core is a key component to several natural products. Herein, we describe the application of a solid-supported Glaser–Hay reaction in the preparation of several natural products. These compounds were then screened for antibacterial activity, illustrating the utility of the methodology

Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1

Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of <i>p</i>-carboxymethyl-l-phenylalanine (<i>p</i>CmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, <i>p</i>-benzoyl-l-phenylalanine (<i>p</i>BpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1’s ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein–protein interactions