Page 23 - Tyrosine-Based Bioconjugations - Jorick Bruins

While sortase A offers an efficient way to enzymatically label some proteins, during ligation the removed C-terminus of an LPXTG-fused protein becomes a substrate for the sortase ligation itself. This reversibility, combined with the fact that water can attack the acyl intermediate to yield the hydrolysis product in an irreversible manner,76 compromises high ligation yields unless a large excess of glycine-bearing probe is used. This in turn means that protein-protein fusion is unfavorable when using sortase ligation due to high stoichiometric amounts of potentially valuable protein and/or solubility issues due to high protein concentrations. To circumvent this, a step-wise approach may be envisioned based on the introduction of click chemistry handles on proteins for subsequent functionalization, which can lead to efficient production of protein- protein conjugates such as bispecific antibodies.77-79
Tubulin tyrosine ligase (TTL) is an enzyme that catalyzes the addition of tyrosine (Scheme 3B), and analogues thereof, to the C-termini of proteins bearing the Tub-tag (VDSVEGEGEEEGEE).80 By introducing bioorthogonal handles such as azides, subsequent click reactions allow for the conjugation of biomolecules of choice via a two-step ligation strategy similar to sortase A, without the issues of reversible reactions and hydrolysis. Later work demonstrated a much broader substrate scope for TTL, including phenylalanine, L-3,4-dihydroxyphenylalanine (L- DOPA), tryptophan, coumarin, and more.81 The introduction of L-DOPA is of particular use, as oxidation with sodium periodate (NaIO4) generates quinones, which are highly susceptible to Michael addition or Diels–Alder modification. These quinones are also regularly found in Nature, generally generated by enzymes called tyrosinases.
1.3. Tyrosinases
Tyrosinase (polyphenol oxidase, EC 1.14.18.1) is an enzyme that is found in a broad number of species ranging from bacteria, fungi, plants to mammals.82 While tyrosinases differ significantly with respect to their sequences, size and glycosylation patters,83 they are similar in their enzymatic activity. The active site consists of six histidine residues coordinating two copper ions, which can perform oxidation by using molecular oxygen. The enzyme can catalyze both the ortho-hydroxylation of mono-phenols such as tyrosine (22) to di-phenols (23), as well as the subsequent oxidation to di-ketones (24) (Scheme 4).82, 84 These di-ketones are called quinones, which due to the ortho-hydroxylation are only present in the form of ortho-quinones. The formation of quinones via tyrosinases play significant roles in nature, such as the formation of melanins in skin pigmentation to protect against UV radiation via a process called melanogenesis,85, 86 as well as the browning of food products, especially in mushrooms, bananas, apples, pears, potatoes, avocadoes and peaches.87, 88
General Introduction
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