Site-specific PEGylation of proteins containing unnatural amino acids

Abstract

Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast. The azido group was used in a mild [3+2] cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein. This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.

Selective chemical protein modification

“…This has led to a wide range of CuAAC reagents now being commercially available for bioconjugation. Indeed, the CuAAC can be performed site-selectively with complete conversion 34 and has been used in many significant applications, such as the generation of PEGylated proteins 87 , the generation of dual PTM glycoprotein mimics due to its orthogonality to existing cysteine chemistry 34,35 , cellular proteomic analysis (BONCAT) 80 , a quantitative method for primary cell proteomics (QuaNCAT) 88 , and the construction of highly-valent protein nanoparticles 89 . Despite this compatibility, the perceived toxicity of copper has led to the exploration of alternative cycloaddition-type reactions.…”

Crosslinking of viral nanoparticles with “clickable” fluorescent crosslinkers at the interface

“…This reaction has been extensively used in materials development [6][7][8][9], polymer synthesis [10,11], dendrimer synthesis [12][13][14][15][16] and drug discovery [17][18][19]. CuAAC reaction serves as a great tool for bioconjugation applications [20][21][22][23][24][25][26][27][28][29], mainly due to (i) facile synthesis and easy incorporation of alkyne and azide moieties into biomolecular frameworks, (ii) compatibility to other functionalities yielding highly specific products, (iii) compatibility to water which is crucial for biomolecular systems, (iv) mild reaction conditions which will help maintaining the properties of biomolecules, and (v) stability of the resulting triazole ring to the hydrolytic cleavage, oxidation and reduction [2,3,9].…”

Click Chemistry: A Powerful Tool to Create Polymer‐Based Macromolecular Chimeras

“…The enhanced physical and pharmacological properties of PEGylated proteins have driven PEGylation to become probably the most studied area of protein bioconjugation. [99,100] Schultz and coworkers [101] compared the PEGylation of a genetically modified superoxide dismutase (SOD) with either an alkyne-appended PEG and click chemistry or the corresponding conventionally utilized PEG NHS-activated esters ( Figure 5A). For this reason, the Trp33 residue, which was known to be highly exposed on the exterior of the protein, was genetically replaced by a non-natural amino-acid similar to Tyr, bearing an azide instead of the hydroxy group.…”

“…(A) Direct [1,3] Huisgen cycloaddition of azidated PEG to an alkyne bearing, genetically modified SOD by the group of Schultz. [101] (B) Glycoprotein mimics using ATRP and click chemistry by Haddleton and coworkers. [90,104] (C) Copper free tandem [3 þ 2] cycloaddition-retro-Diels-Alder reactions reported for the protein HEWL (i) and the peptide sequence GGRGDG (ii) by the group of Nolte.…”