Phosphorylation is one of the most common and important post-translational modifications of proteins in the biological world. In cells, about 1/3 of proteins are thought to be modified by phosphorylation. Phosphorylation modification of proteins is a common and important regulatory mechanism that plays an important role in the regulation of many biological processes (such as signal transduction, gene expression, apoptosis, etc.). The study of phosphorylated proteins and peptides can clarify the mechanism of many biological processes and help people to further understand the nature of life activities.
The phosphorylation modification of peptides mainly refers to the conversion of the side chain hydroxyl groups of serine (Ser), threonine (Thr) or tyrosine (Tyr) residues in the peptide chain into the form of phosphate. Phosphorylation of peptides is an essential tool for studying protein phosphorylation. Many physiological processes mediated by peptides and proteins are accomplished through the addition and removal of phosphates from substrates.
Phosphorylated tyrosine, phosphorylated threonine, and phosphorylated serine derivatives can either be introduced into the polypeptide during synthesis or formed after polypeptide synthesis.
This method is easy to operate and is the main method for phosphorylation modification of a single site in the peptide chain.
Specific steps: phosphorylate the amino acid (Ser, Tyr or Thr) that needs to be phosphorylated in advance and properly protect it, and then condense the phosphorylated amino acid to the designated site of the peptide according to the normal solid-phase peptide synthesis process. Protecting groups can be selectively removed, enabling selective phosphorylation.
This method is suitable for phosphorylation modification at multiple sites in the peptide chain.
After the solid phase synthesis of the peptide is complete, the side chain protecting groups of the amino acids are selectively removed. For Tyr and Thr, amino acids with unprotected side chains can be used directly for the reaction. The side chain protecting group can be removed quantitatively under the condition of 1% TFA/DCM. When this phosphorylation modification method is used, bisbenzyl phosphoramidite and tetrazolium can be used to generate phosphoramidite tetrazole active intermediate, which is connected to the hydroxyl group, and then oxidized under the condition of peroxyacid to generate phosphoryl group to complete the phosphoric acid. chemical reaction.
|MARCKS Peptide (151-175), Phosphorylated
|Phosphorylase Kinase β-Subunit Fragment 420-436
|MAPK/MAK/MRK overlapping kinase (11-20)
|MAPK/MAK/MRK overlapping kinase (32-40)
|MAPK/MAK/MRK overlapping kinase (352-360)