Page 59 - Tyrosine-Based Bioconjugations - Jorick Bruins

the higher reaction rate for cpTCO. However, the short lifetime of the generated quinone favors labelling with the fast reacting cpTCO by reducing side product formation.
Figure 2. Time-resolved SDS-PAGE analysis, based on heavy chain labelling, of cpTCO-PEG-lissamine SPOCQ with Tras[HC]G4Y (lane 1) in the presence of mTyr (lanes 4–11). No reaction is apparent after 3 h in the absence of mTyr (lane 3), while the present of mTyr alone (absence of cpTCO reagent) leads to degradation/oligomer formation (lane 2).
In order to exploit the lack of reactivity of cpTCO for azides, and high reaction rate with ortho- quinones, we envisioned that tandem, dual labelling of an antibody should be accessible by orthogonal reactions of BCN and cpTCO with azide and (exposed) tyrosine, respectively. To test this, we explore the possibility of tandem installation of two different functional moieties onto a glycan-remodeled monoclonal antibody30 by cpTCO–SPOCQ and BCN–SPAAC, executed in either order. We started by the cpTCO–SPOCQ reaction on the Tras[HC]G4Y C-terminus followed by SPAAC modification of azido-glycan with BCN–MMAE (method A), but also checked method B (Figure 3A).
To ensure the same conjugate is obtained via both routes, we used cpTCO–PEG–lissamine and BCN–sulfamide–MMAE (SI) with a cathepsin-cleavable linker (MMAE = monomethyl auristatin E, a potent antimitotic agent that inhibits cell division). In addition, the sulfamide spacer in the BCN unit enhanced solubility and conjugation efficiency.31
Dual Protein Labeling via SPOCQ and SPAAC
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