title: Reactivity of intein thioesters: appending a functional group to a protein.
abstract: The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706-1322, USA.
authors.forename: Jeet
authors.initials: J
authors.lastname: Kalia
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Raines
mesh_terms: D000588:Amines / Q000737:chemistry; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry; D004952:Esters; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry; D047668:Inteins; D007700:Kinetics; D015394:Molecular Structure; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000588:Amines; D001386:Azides; D003432:Cross-Linking Reagents; D004952:Esters; D005452:Fluoresceins; D011506:Proteins; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic