Page 86 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 5
Figure 3. HPLC chromatograms of (A) Tras[LC]G4Y and SPOCQ with (B) BCN-lissamine or (C) TCO-AF568, and (D) competitive SPOCQ between BCN-lissamine and TCO-AF568. It is noted that there seem to be multiple small peaks present in figure 3B; these are degradation products of the excess BCN–lissamine due to exposure to the relatively harsh HPLC conditions (0.1% formic acid at 80 ˚C).
The ability of BCN-SPOCQ to outcompete TCO-SPOCQ paves the way for the selective introduction of TCO-moieties at G4Y sites by using bifunctional linkers that contain both BCN and TCO. To this end, BCN-NHS (4) was subjected to H2N-PEG3-TCO (5) to yield BCN–PEG3–TCO (6). Subsequently, 6 was compared to commercially available BCN–PEG3–BCN (7) in a 2-step conjugation process (Figure 4B). First, SPOCQ with 6 or 7 was performed, after which excess linker was removed by Amicon® centrifugal filters (50 kDa MWCO). Then, methyltetrazine bearing the fluorophore TAMRA (MeTz–TAMRA, Figure S4) was added for inverse-electron
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