Bioconjugation: linkage stability and novel methods
The remarkable stability of amides makes them highly desirable for bioconjugation. We have developed a general method for the site-specific modification of proteins via amide linkages. In this method, proteins are first labeled on their C-termini with the azido functional group by utilizing intein chemistry. Subsequently, a chemoselective amide bond-forming reaction of the azido group---the Staudinger ligation---is performed on the azido-proteins. Using this approach, we have immobilized the ribonuclease A protein rapidly and site-specifically via amide linkages. Furthermore, we have utilized this method to generate azido-single-chain antibodies. Finally, we have improved the scope and the efficiency of this method by integrating it with yeast surface display, to enable the efficient production of both prokaryotic and eukaryotic azido-proteins.
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Hydrolytic Stability of Hydrazones and Oximes
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1,3-dipolar azide-alkyne cycloaddition 13C NMR 1H NMR 25 mM sodium a-hydrazino acetamido acid alkane thiols alkynyl fluorescein 4.10 amide amine aqueous solution Ar(g assays azide azido azido group azido-proteins azido-RNase azido-scFvs azido-sm3E bioconjugation biomolecules biotin Bruice buffer at pH calcd catalyst cell Chem chemistry chemoselective cleavage coli cysteine dissolved disulfide domain fusion protein expressed protein ligation Figure fluorescence functional group HRMS Huisgen 1,3-dipolar azide-alkyne Huisgen 1,3-dipolar cycloaddition hydrazones hydrazones and oximes hydrolysis immobilized RNase incubated intein Jencks Kalia kinetic traces linkages loaded in lane maleimide method microarrays mmol native chemical ligation Nilsson nitrogen nucleophiles NMR 100 MHz overlaid oxime peptide protonation purified by flash rate constant reaction mixture reagent reduced pressure removed under reduced resulting rinsed with 25 RNase room temperature scFv Scheme SDS-PAGE sodium phosphate sodium phosphate buffer Soellner Staudinger ligation succinimide succinimide 3.2 Synthesis thioester thiol utilized wild-type RNase yeast surface display