Page 18 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 1
report describes the use of sulfonyl acrylate reagents to label the most reactive e-amino group of one specific lysine in a selective manner.48 While this method only labels a single lysine residue, it does not allow for targeting of a specific conjugation site. Which can be suboptimal, as different conjugations sites can have varying effects on conjugate functionality and pharmacokinetics.49
Cysteine residues (4) are, due to their nucleophilic thiol residues and low occurrence in proteins (1-2%),45 uniquely reactive in proteins and therefore highly employed for bioconjugation. These cysteine residues are either unpaired, resulting in free thiols, or paired to form disulfide bridges vital for the stability of the protein.50 Generally, thiols can be generated by reducing disulfide bridges, also known as cystines, with reagents such as dithiothreitol (DTT) β-mercaptoethanol (BME), or tris[2-carboxyethyl]phosphine (TCEP).45 Most commonly, the cysteine thiol moiety is selectively be modified via Michael addition with a maleimide reagent (Figure 4B, 5), however a-halogenated carbonyl compounds, vinyl sulfones or other electrophilic agents can also be employed.51 While these modifications are performed easily, maleimide conjugates may suffer from instability issues, while others may be accompanied by side-reactions.45, 52, 53 Moreover, in case multiple cystines are present in a protein, a heterogeneous mixture of conjugates may result.54, 55
Figure 4. (A) Schematic representations of Lysine-NHS conjugation and the resulting random conjugation on antibodies. (B) Schematic representations of Cysteine-maleimide conjugation and the resulting random conjugation on antibodies after disulfide bridge reduction.
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