Protein C-terminal modification through thioacid/azide amidation.
The preparation of protein bioconjugates has been largely dependent on the development of selective chemistries that are orthogonal to the diverse functionalities present in a protein. Here, we report a new method for C-terminus-directed modification of recombinant proteins. The method is based on the thioacid/azide amidation reaction. Essentially, hydrothiolytic cleavage of the thioester intermediate in protein splicing yields a recombinant protein with a unique thioacid group at the C-terminus, which is then chemoselectively amidated with an electron-poor organic azide carrying a biofunctional tag. The small ubiquitin protein was used as a model system to demonstrate the utility of this new bioconjugation method. C-terminal PEGylation or biotinylation of ubiquitin was readily achieved through amidation of ubiquitin thioacid with a sulfonazide-functionalized PEG or biotin derivative. Our data validate that thioacid/azide amidation is a mechanistically novel and practically useful method for site-selective protein modification.
Zhang
Xiaohong
X
Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Li
Fupeng
F
Lu
Xiao-Wei
XW
Liu
Chuan-Fa
CF
D000577:Amides / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013455:Sulfur / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry
D000577:Amides; D001386:Azides; D011506:Proteins; D025801:Ubiquitin; D011092:Polyethylene Glycols; D013455:Sulfur