Page 87 - Tyrosine-Based Bioconjugations - Jorick Bruins

Preparation of Trifunctional Protein-antibody Conjugates
Figure 4. (A) Structures of BCN-PEG3-TCO (6) and BCN-PEG3-BCN (7), and schematic representation of MeTz– TAMRA. (B) Reaction scheme of the two-step conjugation to introduce MeTz-bearing handles, only TCO- bearing antibodies are displayed. (C) SDS-PAGE analysis of AT1005[HC]ST–[LC]G4Y before oxidation (lane A), after mTyr oxidation (lane B), SPOCQ with 2.5 or 5 eq. of bis-BCN 7 (lanes C, D), or SPOCQ with 5 eq. of BCN- TCO 6 (lane E), and AT1005[HC]G4Y–[LC]ST before oxidation (lane F), after mTyr oxidation (lane G), SPOCQ with 5 eq. of 6 (lane H), or SPOCQ with 86, 172 or 344 eq. of 7 (lanes I, J, K).
demand Diels–Alder (IEDDA) with the TCO- or BCN-bearing antibodies (Figure 4B). The two-step conjugation method was tested on AT1005 with either a G4Y on the light chain and sortase on the heavy chain, or vice versa.21 The sortase tag (LPETG) is not expected to perform any reaction
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