Site-specific modification of peptides and proteins is ak ey aspect of protein engineering.W ed eveloped am ethod for modification of the Nterminus of proteins using 1H-1,2,3-triazole-4-carbaldehyde( TA4C) derivatives, which can be prepared in one step. The N-terminal specific labelingo fb ioactive peptides and proteins with the TA4C derivatives proceeds under mild reactionc onditions in excellent conversion (angiotensinI: 92 %, ribonuclease A: 90 %). This method enables site-specific conjugation of various functional molecules such as fluorophores,b iotin, and polyethylene glycol attached to the triazole ring to the Nterminus. Furthermore, af unctional molecule modified with ap rimary amine moiety can be directly converted into aT A4C derivativethrough aDimroth rearrangement reaction with 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde. This methodc an be used to obtain N-terminal-modifiedp roteins via only two steps:1 )convenient preparation of aT A4C derivativew ith af unctional group and 2) modification of the Nterminus of the protein with the TA4C derivative.
Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The Nterminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.
Redox-active cytochromeb562with a tethered azide group on the heme propionate side chain is covalently linked to an acetylene moiety introduced on the sidewall of a single-walled carbon nanotube by copper-catalyzed click chemistry.
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