Development And Application Of Glutamate/aspartate Chemoselective Probes

Chemoselective modification is a powerful method for the study of protein expression and function;Over the years,researchers have developed a series of Chemoselective modification molecules targeting cysteine,lysine,methionine and other amino acid residues.They combined these molecular probes with activity-based protein profiling(ABPP)technology to discover a series of highly active amino acid sites and new drug targets,which provided an important basis for drug discovery and disease treatment.Acidic amino acid residues include glutamic acid and aspartic acid in organisms,which account for 12.1% of the total amino acid residues,especially some acidic amino acid residues play a key role in maintaining protein structure and function.However,due to the relatively weak nucleophilic ability of acidic amino acid residues,it is still a challenging subject to achieve chemoselective modification of acidic amino acid residues in complex organisms.This thesis combined the research foundation of our team on the modification of acidic amino acids,and examined various chemical reactions containing carboxylic acids as reactants.We have found that 3-phenyl-2H-azirine could make intermolecular coupling reaction with carboxylic acid under mild conditions.The carboxylic acid protonates 2H-azirine,then undergoes nucleophilic addition and intramolecular rearrangement to generate a stable N-acetyl amide coupling product.However,other amino acid residues cannot protonate 2H-azirine,so we assume that 2H-azirine could be used as a chemoselective probe of acidic amino acids in vivo.In order to verify this idea,we synthesized a series of molecular probes based on 2H-azirine(AZ-1 ～ AZ-11),mainly introducing alkyl or aryl groups into the 2,3 position,to investigate the effect of different substituents on the labeling efficiency and selectivity of probes.We first tested the chemical stability of the probe under physiological conditions by NMR.The experimental results showed that the probes can be stored in water solution for more than 3 days at room temperature.Then studied the reactivity of AZ-11 with each amino acid side chain by HPLC,through NMR confirmation,the reaction product of AZ-11 and Boc-protected glutamic acid in PBS/THF mixed solution is a stable N-acetyl amide product which is consistent with our assumption.It was verified by HPLC that the reaction between AZ-11 and the peptide could be complete within 2 hours,and LC-MS/MS confirmed that the modified sites of the probe were glutamic acid and aspartic acid,which proved the probe has good labeling efficiency and selectivity.Subsequently,we investigated the labeling of probes in proteins,the labeling experiments showed that majority probes could significantly label recombinant proteins within 1 h and 2.5 μM probe concentration,and the addition of competitor could effectively inhibit the labeling strength of probes,which proved the labeling effectiveness of probes.Cell-level labeling experiments showed that the labeling ability of AZ-9 was significantly stronger than other probes,indicating that the 2-position substituent group could affect the molecular probes to label proteins in cells.In addition,the probe can still significantly label all kinds of proteins at a probe concentration of 2.5 μM within 1 hour,demonstrating the good labeling efficiency of probes at the cellular level.It was proved that molecular probes could selectively label acidic amino acid sites in proteins and living cells by pull-down combined with biological mass spectrometry,which confirmed that 3-phenyl-2Hazirine could be used as the choice of acidic amino acid residues chemoselective modified probe.We found that the sites labeled by probes are mainly located in the protein pockets,and can modify functional glutamic acid residues with high selectivity through protein crystal structure analysis.Combined with iso TOP-ABPP,we have found a series of highly active acidic amino acid sites in breast cancer cells,which provided an important foundation for the development of breast cancer related drugs.It suggested that the new reactive group we developed,3-phenyl-2H-azirine,could be used to construct new covalent small molecule inhibitors.