Page 20 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 1
1.2.2. Mutagenesis
Chemically orthogonal reaction sites can be introduced with uniquely reactive groups. In the case of antibodies, this can be achieved by expressing new cysteine groups via site-directed mutagenesis or insertion of an extra cysteine residue.59, 60 This results in the generation of a protein with a unique single thiol group, which can be selectively labeled with e.g. maleimides (Figure 5). While these methods lead to site-selective conjugation, solvent-accessible thiols are susceptible to dimerization and require pre-treatment before conjugation can be attempted.59, 61 Furthermore, thiol-maleimide conjugates are instable due to e.g. retro-Michael side- reactions,37 which is a major drawback for ADCs as their stability is of paramount importance for a favorable therapeutic index.
Figure 5. Maleimide conjugation of a cysteine-engineered antibody.
To overcome many of the issues associated with conjugation to canonical amino acids, introduction of new, non-canonical amino acids in proteins was developed.62 By re-allocating the amber stop codon to encode a non-canonical amino acid, the genetic code can be expanded to include new amino acids.63 With the addition of a non-canonical amino acid, functionalities with fully orthogonal reactivity can be expressed on proteins. This allows for easy and rapid functionalization of antibodies without any side-reactions or pre-treatments.62
Noteworthy examples of these bio-orthogonal reactions are the strain-promoted alkyne–azide cycloaddition (SPAAC)64 and the inverse-electron demand Diels–Alder (IEDDA) reaction with tetrazines,65 both of which have been reviewed.38, 66, 67 SPAAC revolves around the (3+2)i cycloaddition between azides, such as p-azidophenylalanine (p-AzF, 16), and strained alkynes, such as bicyclo[6.1.0]nonyne (BCN, 17) for rapid and traceless cycloaddition to yield 18 (Scheme 2A).64 The reaction rate of SPAAC is explained by the fact that the alkyne’s SP-hybridized carbon atoms cannot adopt the ideal 180˚ bond angle, resulting in an increase in ring strain.67 This in
i When denoting cycloadditions, round brackets indicate the atoms involved, whereas square brackets indicate the electrons involved. Azide–alkyne cycloadditions are therefore (3+2), [4+2] cycloadditions. Whereas a Diels–Alder is a (4+2), [4+2] cycloaddition. Source: goldbook.iupac.org/terms/view/C01496
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