Synthetic Peptide and Protein Pyrophosphorylation: Development of New Chemical Tools to Study the Signaling Function of the Inositol Pyropphosphates

Abstract

The inositol pyrophosphates (PP-InsPs) are a class of highly phosphorylated small molecule second messengers which help regulate diverse processes in eukaryotes, including telomere elongation, vacuole formation, and cellular phosphate sensing and homeostasis. In mammals, these molecules are essential for normal insulin secretion and adiopgenesis, supporting the hypothesis that they help to coordinate cell signaling networks and metabolism. One mode by which they have been proposed to transduce signals is via a novel posttranslational modification (PTM) known as protein pyrophosphorylation, in which a PP-InsP transfers its high-energy β-phosphoryl group onto the pre-existing phosphoryl group of a protein substrate. However, this modification has only been identified in vitro; in order to elucidate its role in vivo, new chemical tools are needed. Therefore, I undertook the development of methodology for the synthesis of pyrophosphorylated peptide standards. These constructs have since been used to characterize the enzymatic stability and reversibility of pyrophosphorylation, to validate an affinity reagent for the enrichment of pyrophosphorylated peptides, and to develop a mass spectrometry based method for detection and sequencing of pyrophosphopeptides. During the exploration of synthetic methodology, I discovered that a class of phosphorylation reagents known as phosphorimidazolides displayed a striking tendency to react with phosphate monoesters preferentially over other residues found on peptide substrates with rate constants as high as 0.58 M-1s-1 in the presence of Zn2+. A series of phosphorimidazolide reagents incorporating photo-labile protecting groups and affinity tags were synthesized and used to characterize the selectivity of this reaction on full-length proteins. A phosphorimidazolide reagent was used for the selective affinity capture and release of a phosphorylated protein over its non-phosphorylated counterpart.