Page 26 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 1
 Scheme 5. (A) Oxidation of tyrosine to its corresponding quinone, followed by a Michael addition of a nucleophilic side-chain functionality of an amino acid residue R (i.e. Cys, His, or Lys). (B) PNA crosslinking via DOPA-oxidation by NaIO4 and subsequent Michael addition.
1.3.2. Polymerization and cross coupling of proteins
The tendency of tyrosine to polymerize upon exposure to oxidizing conditions finds a prominent role in nature in the form of melanin formation, either through copolymerization with cysteine to form pheomelanin,85 or self-polymerization to eumelanin.100-102 This phenomenon has been widely studied and applied to e.g. grafting of chitosan,103 and immobilization of proteins to amino-modified polystyrene beads.104 Despite the fact that phenolic side-chains of tyrosine residues are themselves incapable of nucleophilic attack on quinone residues,99 Long et al. were able to show that proteins carrying a tyrosine-rich tag (i.e. hemagglutinin-tag, abbreviated as HA-tag) can be used for protein crosslinking upon addition of free tyrosine.105 Interestingly, when the HA-tag (YPYDVPDYA) was expressed at the C-terminus of Escherichia coli dihydrofolate (eDHFR), oxidation of the tyrosine-rich tag by mushroom tyrosinase (mTyr) led to the formation of quinones, which combined with the addition of free tyrosine to the mixture led to cross- coupled HA-labeled eDHFR (Scheme 6). This was due to polymerization of tyrosine, with incorporation of the tyrosine-rich HA-tag. This effect was intensified in case of introduction of a more tyrosine-rich tag (GYGYGYGYGY). Not surprisingly, in the presence of excess nucleophilic amino acids other than tyrosine, functionalization was observed instead of polymerization, whereas fragmentation of the HA-tag was observed in the absence of any additional amino acid.
The use of tyrosine-bearing tags as employed by Long et al. allows for modification on proteins by selective oxidation of tyrosine residues and subsequent functionalization. However, a more selectively addressable coupling strategy is required to prevent non-selective cross coupling between nucleophilic amino acid residue sidechains and the formed quinones.
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