針對蛋白質酪胺酸磷酸水解酶合成分子工具之研究

In light of the growing interest in understanding the roles of protein tyrosine phosphatases (PTPs) in the complicated signaling pathways, convenient methods to profile and analyze PTP activities in various cellular processes are in high demand. This thesis is divided into four parts. In first part, we demonstrate a two-step assay platform that combined the use of a universal activity-based probe with a PTP-specific antibody, by which the designated PTP activity in a cellular event could be successfully determined. Further we describe an improved plate-based assay platform by replacing the immunoprecipitation step with a simpler immunocapture step using an antibody on a plate. An assay protocol targeting SHP2 activity was thus established and a universal probe with a sulforhodamine B tag was used to afford a direct fluorescent readout. In second part, a novel phosphotyrosine analog serving as the latent trapping device has been designed and explored. It allows addition of various amino acid residues to its C- and N-terminus to extend the recognition region. As a proof-of-concept, we have completed the synthesis of three tripeptide probes. These probes all carry the latent trapping device in the middle position and a leucinamide residue at the C-terminus, differing only in their N-terminal amino acid (Glu, Phe, and Lys). The labeling properties of these probes were also determined. The results showed the newly synthesized probes could selectively label protein phosphotyrosine phosphatases (PTPs) in an activity-dependent manner and the amino acid residues flanking the phosphotyrosine analog could help the probes differentiating PTP targets. (S)-4-Hydroxy-3-hydroxymethylphenylalanine is a naturally occurring tyrosine derivative with antihypertensive properties, and the compound is also an important starting material for a variety of synthetic applications. Therefore in third part, we first describe a convenient two-step protocol starting from commercially available Boc-L-Tyr to afford the compound in high yields. Further we synthesize phosphotyrosine mimic device which applies for combinatorial solid phase synthesis with Fmoc strategy. Via change two positions of the designed sequence with seven different amino acids to build up the activity-based probe library of cyclic peptides. In fourth part, we report the design, synthesize, and characterization of new chemical probes that can be used for in vivo labeling of cellular PTPs. The new designed probe has a phosphotyrosine mimic device that is modified to neutralize the negative charges of the phosphate. A non-polar BODIPY is also used as the reported group to help the passage through cell membrane. The newly designed PTP probes effectively enter mammalian cells and label PTPs. Thus our newly designed probes could be used as a tool in detecting and monitoring the cellular activities of PTPs in vivo.