4 research outputs found
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