Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

Abstract

Oxidative coupling (OC) through o-quinone intermediates has been established as an efficient and site-selective way to modify protein N-termini and the unnatural amino acid p-aminophenylalanine (paF). Recently, we reported that the tyrosinase-mediated oxidation of phenol-tagged cargo molecules is a particularly convenient method of generating o-quinones in situ. The coupling partners can be easily prepared and stored, the reaction takes place under mild conditions (phosphate buffer, pH 6.5, 4 to 23 °C), and dissolved oxygen is the only oxidant required. Here, we show an important extension of this chemistry for the activation of tyrosine residues that project into solution from the N or C-termini of peptide and protein substrates. Generating the o-quinone electrophiles from tyrosine allows greater flexibility in choosing the nucleophilic coupling partner and expands the scope of the reaction to include C-terminal positions. We also introduce a new bacterial tyrosinase enzyme that shows improved activation for some tyrosine substrates. The efficacy of several secondary amines and aniline derivatives was evaluated in the coupling reactions, providing important information for coupling partner design. This strategy was used to modify the C-termini of an antibody scFv construct and of Protein L, a human IgG kappa light chain binding protein. The use of the modified proteins as immunolabeling agents was also demonstrated.

Tyrosine bioconjugation – an emergent alternative

“…Although such methods are less time-and cost-effective as a whole due to the preceding preparatory steps, the tyrosinase-catalysed modification of tyrosine-containing tags and subsequent chemical conjugations proceeds quickly and with high conversions under physiological conditions. [49][50][51][52] The ligation of unnatural tyrosine derivatives to proteins has been also successfully achieved. 56 In addition, tyrosine tags are typically small and loss of protein activity was not reported in any of the examples reviewed.…”

“…This is in contrast to the aniline nucleophiles, which add to the 6-position and the products of which exist mainly in quinone form. 51 The cysteine/o-quinone linkage formed was shown to be more stable compared to an analogous thiosuccinimide linkage after incubation in human blood serum for 7 days. 55 The method was used to attach a variety of proteins and peptides to other proteins.…”

“…End35, laminarinase A, trastuzumab fragment) being successfully modified by tyrosinase, with no reaction occurring with the wild type under the same reaction conditions (Scheme 7a). [49][50][51] Although having to genetically modify the protein is generally a downside (e.g. increased cost, time etc.…”

“…compound 28) exhibiting higher efficiency. 51 Therefore, cargo-functionalised anilines were used for tyrosinase activated bioconjugation to a peptide and an antibody fragment. To illustrate the method, a single chain variable fragment (scFv) of trastuzumab bearing a -GGY tag was labelled site-specifically with the fluorescent dye Oregon Green in 1 h with 84% conversion.…”

Tyrosine Conjugation Methods for Protein Labelling

“…The site‐specific oxidation of phenol from Tyr may also be successfully achieved by using mushroom tyrosinase enzyme [36, 37] . The formation of reactive ortho ‐quinone was used for coupling reactions with secondary amines or anilines, [38] boronic acid, [39] cycloalkynes for strain‐promoted cycloaddition (SPOCQ) [40] and mammalian catechol‐ O ‐methyltransferase for O ‐alkylation [41] . The reactivity of ortho ‐quinone species was recently reviewed by van Delft and co‐workers and will be not further developed here [24] …”