Phosphate-Modified Nucleotides for Monitoring Enzyme Activity

Abstract

Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis

“…The conversion of 18 and 19 into their parent natural MSN was achieved by applying an aqueous solution of 18 and 19 to a Dowex-H + plug filtration. This slightly acidic environment smoothly induced P–N-bond hydrolysis, affording pppApp ( 4 ) and pppGpp ( 3 ). After precipitation, 3 and 4 were isolated in total yields of 26% and 18% with respect to the starting materials 7 and 8 after eight and nine steps and only two intermediates that required purification.…”

The Hitchhiker’s Guide to Organophosphate Chemistry

“…Broader discussions of these and adjacent fields can be found in a recent volume of topics in current chemistry. 73,[90][91][92][93][94][95][96] The last years have seen tremendous advances in phosphorylation techniques and phosphate chemistry still provides ample opportunities regarding challenging synthetic targets, method development, and improvement of analytical techniques. As highlights coming from my laboratory, the stereoselective synthesis of 1,5-(PP) 2 -InsP 4 (4) and Ap 3 Th (42) show that given the methodologies now available a good advice when facing highly congested, charged, and unstable organophosphates is: 'don't panic'.…”

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications

“…161 For more applications of phosphatemodified nucleotides, Hacker and co-workers published a review focusing on protein activity investigation with various analytical techniques in multiple biological systems including the use of nucleotides with longer phosphate chains to study polymerase activity and for next-generation sequencing. 162…”