Page 127 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Inducible, Selective Labelling of Proteins via Enzymatic Oxidation of Tyrosine
of oxygen, tyrosine is first oxidized to the corresponding 3,4-dihydroxyphenyl derivative, and subsequently to the ortho-quinone (Figure 2). Since the active site of mushroom tyrosinase is embedded in a large protein structure (120-130 kDa), endogenous tyrosine residues are typically not a suitable substrate for mTyr, resulting in selectivity for exposed tyrosine residues, such as the one located on an engineered G4Y-tag. While the G4Y-tag can be fused conveniently to the protein’s C-terminus as such, it is recommended to add an additional amino acid in case of N- terminal fusion (e.g. methionine, leading to MYG4) as the in situ generated quinone may otherwise undergo intramolecular nucleophilic addition of the terminal amino group, thereby forming a stable 5-membered ring.9
Figure 2. Oxidation of the side-chain of a C-terminal tyrosine residue under the action of mushroom tyrosinase (mTyr) in the presence of oxygen provides ortho-quinone in two consecutive steps.
The electrophilic nature of quinones and their tendency to undergo Michael addition has been widely reported.9 For example, the free thiol residue of cysteine can react rapidly with ortho- quinones, which also applies to amino groups present on lysine side-chain or at the protein N- terminus, or the imidazole ring of histidine. Obviously, any such nucleophilic addition must be fully nihilated or outcompeted in case SPOCQ is desired.9
As mentioned above, strained unsaturated C-C bonds, like the triple bond in BCN and the double bond in cpTCO, readily undergo SPOCQ (Figure 1).2, 3 It must be noted that an additional advantage of BCN is its ability to rapidly undergo cycloaddition with azides in a process known as SPAAC (strain-promoted alkyne-azide cycloaddition), in contrast to cpTCO which is inert to azides.10 As a result, dual labelling of proteins containing both an azide (via SPAAC) and an exposed tyrosine (via SPOCQ) is also possible.3 However, the scope of this chapter is limited to the use of SPOCQ for protein conjugation, as this method allows for easy, selective and nearly quantitative introduction of a range of functionalities of choice based on the canonical amino acid tyrosine. Here, we exemplify the usage of SPOCQ by modifying anti-HER2 antibody trastuzumab with BCN- or cpTCO-bearing fluorophores by oxidizing C-terminal tyrosine residues with mTyr.
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