A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well‐Defined Protein–Protein Conjugates

Abstract

Bioorthogonal chemistry holds great potential to generate difficult‐to‐access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DAinv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin‐based targeted cancer therapies.

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction

“…21 Therefore, this method has a unique advantage for labeling membrane protein complexes, such as GPCR-arrestins, and exploring their dynamic properties. 22 Moreover, bioorthogonal reactions have been developed extensively, including the Cu(I) mediated azidealkyne click reaction, 23 strain-promoted azide-alkyne click reaction, 24 Diels-Alder reactions, 25,26 inverse electron-demand Diels-Alder cycloaddition, 25,27 ketone/aldehyde and hydrazine/ hydroxylamine reaction, 28 "π-clamp" reaction, 29 methionineoxaziridine ReACT reaction, 30 palladium catalyzed bioorthogonal reaction, 31 photo-click reaction 32 and others (Table S2 †). Although these bioorthogonal reactions are extremely valuable, a more user-friendly protein labeling method generating homogenous products with high reaction rate without the usage of acidic pH and metal catalysis will further advance the application of smFRET in investigating membrane protein complexes.…”

Enzymatic Methods for the Site-Specific Radiolabeling of Targeting Proteins

“…For example, Plaks et al demonstrated that upon derivatization of the protein with an azide containing molecule at the level of the LAP tag, it is possible to efficiently chemically label the protein with poly(ethylene glycol) of 5 kDa, with a mannose moiety or with the fatty acid palmitic acid or to immobilize the protein [111]. In a recent paper, Wombacher and co-workers [118] tested a large panel of carboxylic acids containing dienophile and diene scaffolds for bioorthogonal cycloaddition reactions as substrates for the lipoic acid ligase mutant W37V. They selected the best performing substrates in terms of efficiency of ligation by LplA W37V and of performance in the cycloaddition reactions.…”

Protein–Antibody Conjugates (PACs): A Plug‐and‐Play Strategy for Covalent Conjugation and Targeted Intracellular Delivery of Pristine Proteins

“…Furthermore, actively targeted delivery of the protein therapeutics into specific cells is also highly desired to improve the bioavailability at the lesion. Learning from ADCs, proteins can be simply conjugated to an antibody as a fusion protein [23–31] . Note however that one of the limitations of ADCs is their low drug‐to‐antibody ratio of ≈4 in most formulations.…”

“…Learning from ADCs, proteins can be simply conjugated to an antibody as a fusion protein. [23][24][25][26][27][28][29][30][31] Note however that one of the limitations of ADCs is their low drug-toantibody ratio of % 4 in most formulations. With the antibodyprotein fusion analog, this is even more amplified, as a reasonable protein-to-antibody ratio (PAR) would be just 1.…”