Page 60 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 3
Figure 3. Schematic depiction of the two routes for dual labelling of antibodies with lissamine and MMAE. Method A: modification of the G4Y-tag using the cpTCO–SPOCQ reaction with cpTCO–lissamine, followed by SPAAC reaction with BCN–MMAE. Method B: employs first a SPAAC reaction on the azido-glycan followed by SPOCQ on the G4Y-tag of the heavy chain.
Thus, clean and efficient enzymatic glycan remodeling was achieved by endoglycosidase and glycosyltransferase, to install 6-azido-GalNAc onto the core-GlcNAc of transiently expressed Tras[HC]G4Y (Figure 3A and Figure S13). Next, a SPOCQ reaction between cpTCO–lissamine and the G4Y-tag on the antibody heavy chain was performed in the presence of mTyr (Figure S14). The observed mass of 25768 Da (Figure 3B, yellow peak) corresponds to the SPOCQ product of the azido-glycan antibody and cpTCO–lissamine (expected: 25766 Da), and corroborates the complete lack of reactivity of the cpTCO moiety with the azido-group on the glycan chain. Subsequently, SPAAC modification of the azido-functionalized glycan chain with BCN–MMAE resulted in the clean formation of the dual labelled fluorescent antibody–drug conjugate. HPLC analysis showed almost complete conversion (Figure S15), and MS analysis identified the presence of the target compound with a FW of 26516 Da (expected: 27275 Da) (Figure 3B, green peak in left spectrum). The difference between the observed an expected mass corresponds to loss of the C-terminal part of MMAE due to fragmentation in the mass chamber (∆762 Da).
Lastly, the tandem labelling steps were performed in reverse order, i.e. first SPAAC reaction on the remodeled azido-glycan chain using BCN–MMAE SPAAC (Figure S16), then cpTCO– lissamine SPOCQ reaction on the G4Y-tag upon tyrosinase oxidation (Figure S17). As expected, the same final product is obtained, albeit via a different intermediate product. We were happy to find that in this case, besides the mass of the linker-fragmented final conjugate (at 26514 Da), the mass of the desired dual labelled ADC is also observed at 27279 Da (expected: 27275 Da) (Figure 3, right set of MS spectra). This difference is presumably due to some experimental variability during the MS analysis.
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