CN-121991149-A - Adenosine phosphate photoaffinity probe and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection and analysis, and particularly relates to an adenosine phosphate photoaffinity probe, a preparation method and application thereof. The probes comprise an AMP-N photoaffinity probe, an ADP-N photoaffinity probe and an ATP-N photoaffinity probe, wherein a photoaffinity group biaziridine with a small volume and an alkynyl group capable of performing biological orthogonal reaction are modified at the phosphate position of adenosine phosphate, and a connecting bond capable of being cracked under acidic conditions is designed between the adenosine phosphate and a photoreactive group. The probe has high stability and good labeling efficiency, can specifically label the adenosine phosphate binding protein and identify the binding site thereof, is suitable for screening, functional research, affinity analysis and discovery and verification of related drug targets of the adenosine phosphate binding protein, and has wide application prospect.

Inventors

• CAI RONG
• GAO CAN
• WANG LUPING

Assignees

• 山东大学

Dates

Publication Date

20260508

Application Date

20260129

Claims (10)

1. 1. An adenosine phosphate photoaffinity probe, wherein the photoaffinity probe has the following structure: 。
2. 2. The method for preparing the photoaffinity probe according to claim 1, wherein the method comprises the following synthetic route: 。
3. 3. a kit comprising at least the photoaffinity probe of adenosine phosphate of claim 1.
4. 4. The kit of claim 3, further comprising a buffer solution and a reaction reagent.
5. 5. Use of the adenosine phosphate photoaffinity probe of claim 1 or the kit of any of claims 3 to 4 in a phosphoadenosine binding protein related study.
6. 6. The use according to claim 5, wherein the use comprises an adenosine phosphate binding protein labelling and/or fluorescent gel imaging assay.
7. 7. The use according to claim 5, wherein the use comprises the identification of an adenosine phosphate binding protein binding site.
8. 8. The use of claim 5, wherein the use comprises an adenosine phosphate binding protein affinity assay for nucleoside phosphates.
9. 9. The use according to claim 5, wherein the use comprises analysis of novel adenosine phosphate binding proteins and related drug targets.
10. 10. The use according to claims 5-9, wherein the cell in which the adenosine phosphate binding protein is located is a prokaryotic cell or a eukaryotic cell, the prokaryotic cell is a bacterial cell, the eukaryotic cell is a fungal cell, a plant cell or an animal cell, the animal cell is further a mammalian cell, and further a HEK293T cell; The adenosine phosphate binding proteins include ATP binding proteins, ADP binding proteins, and AMP binding proteins.

Description

Adenosine phosphate photoaffinity probe and preparation method and application thereof Technical Field The invention belongs to the technical field of biological detection and analysis, and particularly relates to an adenosine phosphate photoaffinity probe, a preparation method and application thereof. Background The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art. Adenosine phosphates such as Adenosine Monophosphate (AMP), adenosine Diphosphate (ADP) and Adenosine Triphosphate (ATP) play a key role in a variety of biological processes such as energy metabolism, signal transduction, biosynthesis and post-translational modification of proteins. Among them, ATP is used as "energy currency" in cells, and its hydrolysis to ADP and inorganic phosphate (Pi) provides driving force for numerous biosynthetic pathways and cellular activities, such as muscle contraction, nerve impulse conduction, mass transport, biosynthesis, and the like. In addition, ATP can also be used as a signal molecule to participate in cell communication, and extracellular ATP can activate purinergic receptors on the cell surface to trigger intracellular signaling cascades, thereby regulating cell growth, differentiation, apoptosis and immune response. ADP also has important physiological functions in organisms. It is both the "precursor" of ATP and its "hydrolysate" and the interconversion between the two forms the core of the energy cycle of the cell. The concentration change of ADP is a key signal for the cell to perceive the energy state. The high concentration of ADP can activate metabolic pathways such as glycolysis, oxidative phosphorylation and the like, and promote the regeneration of ATP. AMPs are also core molecules in the energy metabolism process, involved in energy storage and transfer, signal transduction, and metabolic regulation. The ratio of intracellular ATP to AMP concentration is typically maintained at about 100:1, and changes in this ratio reflect dynamic changes in the energy state of the cell. The biological function of adenosine phosphate is mainly achieved by interactions with its binding proteins, which contain various classes such as kinases, atpases, chaperones, transporters, structures and motor proteins, which play important roles in signal transduction, DNA replication and repair, protein synthesis and folding, intracellular trafficking, cell motility, etc. The imbalance of ATP binding proteins can lead to a variety of diseases, including cancer, cardiovascular disease, metabolic disease, and neurological disease, and therefore they are also important drug targets. ATP binding proteins are also typically capable of binding ADP, except that the affinity of binding may be different from ATP. Although AMP-binding proteins also have important biological functions, less research is directed to AMP-binding proteins than ATP-binding proteins. Systematic studies on these adenosine-phosphate proteins will help elucidate their regulatory mechanisms, analyze the mechanism of action of the relevant drugs, and drive the development of new therapies. The chemical proteomics method adopts a labeling and enrichment technology based on a probe, and combines high-resolution liquid chromatography-mass spectrometry analysis, so that the ATP binding protein can be systematically analyzed on the scale of a proteome. The previously reported ATP acyl phosphate probes are capable of transferring biotin or desulphated biotin groups to lysine residues near the ATP binding pocket, thereby allowing enrichment and analysis of the labeled protein. These probes show good efficiency in identifying ATP binding sites and have been widely used in kinase-related assays, however, their labeling is limited to lysine residues and the probes are less stable. In order to improve the stability and the labeling efficiency of the probe, the applicant previously developed an ATP-O photoaffinity probe which is provided with a photoreactive group biaziridine on gamma-phosphate of ATP, can covalently label ATP-binding proteins under ultraviolet irradiation, contains a bio-orthogonal group alkynyl, and can perform enrichment or fluorescence analysis on the labeled proteins. The probe has been successfully applied to the high-efficiency analysis of ATP binding proteins and can be used for target recognition of kinase inhibitors. However, the large protein modifications in this approach prevent the identification of probe-labeling sites. Disclosure of Invention Aiming at the defects of the prior art, the invention provides an adenosine phosphate photoaffinity probe, and a preparation method and application thereof. Specifically, the invention provides a novel ATP-N photoaffinity probe, which can reduce the modi