Page 84 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 5
The potential to create trifunctional antibodies by chemoenzymatic modification based on the combination of SPOCQ and sortase ligation allows for the modular preparation of trifunctional antibodies without the need of antibody reengineering. Here, we describe a two-step conjugation method for efficient post-recombinant antibody functionalization based on the combination of strain-promoted chemistries. After selection of the correct probe(s) for this two- step conjugation, we optimized the procedure for conjugation with scFvs, cytokines, and oligonucleotides. Finally, the method was combined with sortase A conjugation strategies to create trifunctional antibodies with various valencies.
5.2. Selection of a bifunctional linker
In chapter 4 we discussed the possibility of performing SPOCQ with BCN–UCHT1 on antibodies. It was found that knob-in-hole antibodies can undergo mono-functionalization in a clean and rapid fashion,9 however symmetrical AT1002[HC]G4Y does not perform SPOCQ to full completion (Figure 2). One potential explanation is that after conjugation of BCN–UCHT1 to one of the heavy chains, the second G4Y site becomes inaccessible for mTyr to oxidize, preventing a second cycloaddition from occurring.
Figure 2. (A) Schematic representation of SPOCQ between BCN–UCHT1 and Tras[HC]G4Y, and (B) SDS- PAGE gel of SPOCQ between BCN–UCHT1 and Tras[HC]G4Y.
To ensure selective and efficient protein–protein conjugation, we opted to apply SPOCQ to introduce an elongated linker with a terminal chemical handle for subsequent protein–protein functionalization. To this end, a bifunctional linker was envisioned with an azide on one end and the strained alkene cpTCO (cyclopropanated trans-cyclooctene) on the other end, based on the assumption that azide and cpTCO are mutually inert,20 by cpTCO-NHS (1)20 and a PEG-bearing azide (2). However, synthesis of the cpTCO–azide (3) consistently gave no product, and 1H-NMR showed the disappearance of the alkene peaks at 5.1 and 5.8 ppm after conjugation of cpTCO-
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