Page 56 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 3
3.1. Introduction
Bioorthogonal chemistry can be defined as the combined arsenal of reactions between two (or more) unique molecular functionalities that together rapidly form a covalent bond, but are inert to the remaining repertoire of chemical groups present in the surroundings.1-3 As a consequence of this high functional group compatibility, bioorthogonal reactions have inter alia been broadly applied for detection, immobilization and functionalization of biomolecules, such as carbohydrates, nucleic acid and proteins.4 For example, the Staudinger ligation5 paved the way for the selective imaging of cell-surface azidosugars, azide–alkyne click chemistry is a well- established tool in the field of DNA/RNA origami,6 and oxime ligation7 has proven a powerful technology for protein modification.8 Arguably the most powerful bioorthogonal reactions are those defined by metal-free, strain-promoted cycloadditions of alkynes or alkenes,9 of which stand out the strain-promoted azide–alkyne cycloaddition (SPAAC)10 and the inverse electron- demand Diels–Alder cycloaddition (SPIEDAC)11 of strained alkenes and tetrazines.
Conveniently, it has been established that with judicious choice of functionalities, a next level of orthogonality can be achieved, involving mutually exclusive, bioorthogonal reactions proceeding in tandem or even concurrently.12, 13 Besides inherent orthogonality in reactivity, or the application of targeting agents,14 the use of external stimuli to induce bioorthogonal reactivity with temporal and spatial control has also been described, mostly based on photolysis of tetrazole12 or cyclopropenone13 to nitrile imides or cyclooctyne derivatives, respectively. We,15 and others,16 recently described that enzymatic oxidation of aromatic rings can also be used for temporally controlled bioorthogonal chemistry. Specifically, we demonstrated that strain- promoted oxidation-controlled ortho-quinone cycloaddition (SPOCQ) with cyclooctyne,17, 18 can be successfully applied for the site-specific conjugation of a cyclooctyne-functionalized fluorophore or a toxic payload to tyrosine-engineered proteins, including the monoclonal antibody trastuzumab,15 as well as for highly efficient and complete surface modification.19 Interestingly, with these developments, tyrosine is emerging as a very versatile amino acid for various peptide20 and protein21 derivatization strategies.
With the present study, we have expanded the arsenal of SPOCQ chemistry by demonstrating that besides cyclooctynes strained alkenes also display high reactivity for ortho-quinones. Specifically, we show that cyclopropanated trans-cyclooctene (cpTCO)22 rapidly reacts with ortho-quinones (a.k.a. 1,2-quinones),23 enabling clean and efficient tyrosinase-mediated protein conjugation. In addition, the inertness of cpTCO for azides provides a next level of dual, orthogonal bioconjugation, which may be executed without additional purification steps in between reactions.
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