Page 85 - Tyrosine-Based Bioconjugations - Jorick Bruins

Preparation of Trifunctional Protein-antibody Conjugates
NHS with an azide-bearing PEG-moiety (SI S5.1). Subsequent testing revealed that the alkene peaks of cpTCO disappear within hours when an organic azide was present in the solution (SI S5.2).
Scheme 1. Synthesis of bifunctional linkers cpTCO-PEG3-azide (3) and BCN-PEG3-TCO (6).
As an alternative, we considered to replace cpTCO with its less reactive TCO, which undergoes (4+2) cycloaddition with quinones, but TCO would be too slow for efficient conjugation (i.e. 100- fold lower reaction rate than BCN).20 Therefore, we reasoned that a chemoselective reaction would be feasible for a bifunctional linker with BCN on one end (for SPOCQ, i.e. cycloaddition with quinone) and TCO on the other end (for ligation with tetrazine-functionalized protein). To confirm this assumption, Tras[LC]G4Y was treated with mTyr in the presence of BCN-bearing fluorophore lissamine (BCN–lissamine), or TCO-bearing fluorophore AF568 (TCO–AF568). In contrast to our expectation, both BCN–lissamine (Figure 3B) and TCO–AF568 (Figure 3C) gave clean and full conversions to the fluorophore-bearing product, which for TCO had not been demonstrated before. These findings were later confirmed by SDS-PAGE, where a fluorescent band was formed after SPOCQ with TCO–AF568 (Figure 6), and by LC-MS, where the mass increase corresponding to oxidation and subsequent cycloaddition were observed (Figure S7, S13). However, in line with the significant reaction rate difference between BCN and TCO, when SPOCQ was performed by subjecting Tras[LC]G4Y to an excess of a 1:1 mixture of BCN–lissamine and TCO–AF568, only the BCN-SPOCQ product was detected (Figure 3D).
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