Page 61 - Tyrosine-Based Bioconjugations - Jorick Bruins

Dual Protein Labeling via SPOCQ and SPAAC
Figure 4. The deconvoluted mass spectra of the different steps of the tandem bioconjugation process using the two methods are shown. The main peak in the black trace identifies with GlcNAc-(1,6-Fuc)-6-N3-GalNAc, the minor peak at ∆ -145: GlcNAc-6-N3-GalNAc; in-line fragmentation of the MMAF toxin is also visible.
3.3. Conclusion
In conclusion, we have uncovered a useful addition to the toolbox of copper-free click chemistry, i.e. the rapid reaction of strained alkenes and ortho-quinones, and used this reaction to establish a convenient and high-yielding new strategy for the labelling of antibodies. The rate of the SPOCQ reaction between cpTCO and ortho-quinone is approximately 2.5 times higher than that for BCN, approximately 300 times higher than that for TCO, and >103 times higher than that for cyclopropene; the last two strained alkenes would be too slow for efficient SPOCQ labelling of proteins, due to the high reactivity of the quinone, e.g. with competing nucleophiles. This high reactivity of cpTCO enables faster and cleaner conjugation, as superior labelling of a G4Y- derivatized antibody with a fluorescent moiety was demonstrated when compared to its BCN counterpart. Ultimately, we developed a convenient protocol for the dual labelling of an antibody with a toxic payload and a dye using the orthogonal character of cpTCO and BCN, which may find application in the assessment of internalization potential of glycan-conjugated antibody–drug conjugates for targeted therapy.31 Our setup allows a convenient preparation of a glycan-remodeled azido-containing antibody that also contains a peptidic G4Y tag, and to use this in a two-step dual-labelling strategy to obtain homogeneous ADCs. We note that other methods have been reported for metal-free click conjugation to unnatural amino acids genetically encoded into monoclonal antibodies, for example p-acetylphenylalanine32 or p- azidomethylphenylalanine,33 followed by oxime ligation or SPAAC, respectively. However, low antibody titers (1 g L-1 maximum), which are significantly lower than expression titers on monoclonal antibodies based on canonical amino acids alone (3-7 g L-1 ), limit such applications. Similar arguments apply to genetic encoding of L-DOPA into proteins, which is also suitable for SPOCQ modification.34 We expect that our current method will find widespread application, not only in the preparation of ADCs but extending to other applications in the field of bioconjugation,
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