Expanded Click Conjugation of Recombinant Proteins with Ubiquitin‐Like Modifiers Reveals Altered Substrate Preference of SUMO2‐Modified Ubc9

Abstract

Wrestling with SUMO: the chemical conjugation of proteins with small ubiquitin-like modifiers (SUMO) can be achieved by a copper(I)-catalyzed cycloaddition and unnatural amino acid mutagenesis. This approach overcomes previous restrictions related to the primary sequence of proteins and coupling conditions. Moreover, biochemical data suggests that this triazole linkage presents the modifier in a proper distance and orientation relative to the target protein.

Polystyrene-supported ionic liquid copper complex: A reusable catalyst for one-pot three-component click reaction

“…Based upon the premise, organic synthesis should take advantages such as being modular, stereospecific, high-yielding and involving simple and green protocols [8]. Preparation of 1,2,3-triazoles by copper-catalysed alkyne-azide cycloaddition (CuAAC) has emerged as a prominent "Click" chemistry [9][10][11]. 1,2,3-Triazoles have received considerable attention in synthetic organic chemistry due to their numerous biological and pharmaceutical activities such as anti-HIV [12][13][14], antibacterial [15][16][17], and antiallergic [18] properties.…”

Synthesis of Heterobifunctional Protein Fusions Using Copper‐Free Click Chemistry and the Aldehyde Tag

“…[3,14,15] An alternative approach to generating protein-protein fusions is through chemical conjugation. Native chemical ligation of C-terminal thioesters with b-amino thiols is a powerful method for generating protein-protein fusions, [16][17][18] but at least one coupling partner must be linked at its terminus. In principle, greater topological diversity can be achieved by introducing bioorthogonal functional groups at specific amino acid side chains of the two proteins.…”

Chemical Methods for Protein Ubiquitination

“…global amine protection with Cbz-Osu sumoylated proteins [36] (Scheme 14). In their approach, an alkyne group was introduced onto the C-terminus of ubiquitin(1-74) or ubiquitin-like proteins (such as SUMO) through reacting propargyl amine with the ubiquitin or SUMO thioester.…”

“…The limitations of the method are that the linkage is much longer than the native lysine side chain and that the disulfide bond is also susceptible to reducing conditions. Triazole-linked ubiquitin conjugates were also demonstrated in the synthesis of proteins modified by ubiquitin or ubiquitin-like proteins (Schemes 13 and 14) [34][35][36]. The triazole linkage was formed through copper-catalyzed click reaction between an alkyne and an azide.…”

“…The triazole linkage was formed through copper-catalyzed click reaction between an alkyne and an azide. Both the alkyne and azide are non-natural functional groups and have been introduced through genetic incorporation as part of the unnatural amino acids [34] or through post-expression chemical manipulation of recombinant proteins [35,36]. In the genetic incorporation approach, an azide functional group was introduced when azidohomoalanine was incorporated into the donor ubiquitin at the C-terminal end.…”