Page 33 - Tyrosine-Based Bioconjugations - Jorick Bruins

SPOCQ was used to conjugate on proteins via the introduction of non-canonical amino acids BCN–lysine (Figure 7B, 47) and L-Dihydroxyphenylalanine (Figure 7C, 50).128, 132 With the BCN– bearing proteins, SPOCQ could be achieved be adding quinones (48) bearing fluorescent tags within a few minutes.128 This approach however exposes nucleophilic amino acids such as lysine, cysteine, and histidine to quinones prone to Michael addition, resulting in unwanted side- reactions. The alternative is to introduce L-dopa by virtue of non-canonical amino acid (50) and perform the cycloaddition by oxidizing the diol with sodium periodate,132 the generated quinone (51) is then available to perform SPOCQ with BCN-bearing probes (52). While this method works and yields clean conjugates, the use of non-canonical amino acids is suboptimal due to aforementioned decrease protein titers.
1.5. Outline of this Thesis
The research described in this thesis centers around the chemoenzymatic, site-specific modification of proteins, in particular monoclonal antibodies, by the combination of (a) introduction of an exposed tyrosine residue, (b) oxidation of tyrosine side-chain by mushroom tyrosinase (mTyr) to quinone, allowing for (c) a one-pot conjugation strategy based on strain- promoted cycloaddition of strain ring to the resulting quinone (SPOCQ).
In chapter 2, we describe the concept of employing SPOCQ for site-specific labeling of an engineered protein based on C-terminal fusion of a tetra-glycyltyrosine tag (G4Y-tag) based on tyrosinase-mediated quinone generation and reaction with cyclooctyne, and application of the new methodology for the generation of fluorescently labeled antibodies and antibody-drug conjugates (ADCs). In chapter 3, we demonstrate that SPOCQ can also be achieved with a strained alkene, i.e. cyclopropanated trans-cyclooctene (cpTCO), and its orthogonality with SPAAC, thereby allowing the generation of dual-functionalized antibodies by performing two reactions in tandem. In chapter 4, it is described how knob-in-hole (KiH) technology can be applied to introduce a single molecule (or protein) of interest on an antibody with either SPOCQ or sortase ligation. In chapter 5, we demonstrate a two-step conjugation method for rapid and selective protein-protein conjugation by combination of sortase ligation and SPOCQ, and use the combination of various technology to generate trifunctional antibodies-protein conjugates, bearing scFvs and cytokines. In chapter 6, we report on SPOCQ performed on tyrosine residues residing in solvent-exposed loops residing in an internal sequence of engineered antibodies. Chapter 7 contains an overarching discussion of the work reported in this thesis, give a detailed overview of performing SPOCQ on a technical level, and we examine future possibilities of SPOCQ on proteins. Finally, the appendix contains a step-by-step protocol for performing SPOCQ on proteins.
General Introduction
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