Page 22 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 1
820 M-1 s-1.69 While many, faster reacting, trans-cyclooctenes exist,38 regular TCO is a widely used for protein labeling experiments due to its stability and commercial availability. TCO moieties can be expressed on proteins by introducing e-trans-cyclooct-4-ene lysine (TCO-K, 19), allowing for labeling with tetrazine-bearing compounds to yield stable conjugates (Scheme 2B, 20).
1.2.3. Enzymatic ligations
As an alternative to the use of non-canonical amino acids, labeling of proteins can be achieved via enzymatic modification of peptide tags.71 This approach can circumvent issues with the introduction of non-canonical amino acids, such as low protein titers that generally accompany those techniques.10 By using enzymes, catalysis of reactions can be applied in high specificity under mild conditions on a specific amino acid sequence. This approach is ideal for protein labeling, as this allows for site-selective modification under physiological conditions.
Many examples of enzymatic protein labeling, all of which have been reviewed.71, 72 One of the most commonly applied enzymes for protein modification employs sortase A, a bacterial Ca2+- dependent transpeptidase. Sortase A recognizes the sequence LPXTG, where X can be D, E, A, N, Q, or K (E is used most often).73 Sortase cleaves the amide bond between T and G residues using the highly reactive thiol in its active center and forms an intermediate thio-ester with the LPXT- bearing protein. Subsequently, the free amino group of an N-oligoglycine-terminated peptide or protein can act as a nucleophile, and yield a new ligated peptide bond (Scheme 3A). Generally, C-terminal LPETG peptide tags are fused to the N-terminus of protein, there are however also procedures describing N-terminal sortase ligation74 and even sortase ligation in internal protein loops.75
Scheme 3. Schematic representations of (A) the sortase reaction, and (B) the tubulin tyrosine ligase.
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