Page 58 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 3
Figure 1. Determination of the reaction rate constant of quinone 1 (50 μM) with cpTCO 2 (100 μM) in H2O:MeOH (1:1). Three separate experiments are depicted in the graph.
After this, we explored the suitability of cpTCO-quinone cycloaddition for protein conjugation (Figure 2 and Figure S6–S9). To this end, a cpTCO-functionalized lissamine–PEG conjugate was synthesized and compared head-to-head to its BCN counterpart in labelling of a model antibody (trastuzumab), which contained an exposed G4Y-tag on each heavy chain (abbreviated as Tras[HC]G4Y). Previously, we already showed that without this G4Y-tag, labelling of a smaller protein or an antibody did not proceed.15 In analogy to our previous work, we studied the alkene–SPOCQ reaction between cpTCO–PEG–lissamine and Tras[HC]G4Y upon incubation with mushroom tyrosinase (mTyr) to oxidize the exposed tyrosine phenol ring of the G4Y-tag to ortho- quinone.23 Indeed, when Tras[HC]G4Y was mixed with mTyr in the presence of cpTCO–lissamine, rapid labelling of the antibody was observed that apparently levelled off after approximately 90 minutes (Figure 2, lanes 4–11); no labelling was observed in the absence of mTyr (Figure 2, lane 3). In the absence of a G4Y-tag on the light chain, labelling of this part of the antibody was not observed (i.e. no fluorescent bands around 25 kDa were observed), thus demonstrating (1) the bioorthogonality of alkene–SPOCQ, and (2) the need for a G4Y-tag on the protein. Based on HPLC analysis, a yield of approximately 95% may be calculated (Figure S10–S12), with reduced side- product formation in comparison to alkyne-SPOCQ (Figure S9), i.e. antibody oligomers formed by cross-linking of the ortho-quinone with endogenous protein nucleophiles (Figure 2, lane 2). The reduced formation of protein oligomers for cpTCO versus BCN further corroborates the higher reaction rate with ortho-quinones. Since oxidation of tyrosine residues by mTyr is the rate-limiting step in either SPOCQ reaction,24 the total reaction time does not decrease despite
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