Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction

Abstract

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective tri-functionalization of proteins is readily achieved. In particular cases, we noted PTAD decomposition resulted in formation of a putative isocyanate by-product that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene) glycol chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.

Antibody Conjugates: From Heterogeneous Populations to Defined Reagents

“…By using cyclic diazodicarboxamide derivatives such as 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD), Ban et al established an efficient click-like tyrosine ligation and successfully conjugated PTAD-functionalized substrates to trastuzumab [67,68]. Recently, they described a novel approach to conjugate via tyrosine residues in which diazonium hexafluorophosphate reagents are used to selectively modify trastuzumab.…”

How Far Are We from Dose On Demand of Short-Lived Radiopharmaceuticals?

“…This structure reacts in a reversible way (using different temperatures, Fig. 6.2) with indoles through an ene click reaction [45]; however, it undergoes fast Diels-Alder reactions with dienes and is widely used in biology for its capacity to bind irreversibly tyrosine residues [46]. Such an approach could be very useful in DOD processes aimed at protein labeling, for which radiolabeled prosthetic groups enabling click chemistry are now widely employed [47].…”

Synthesis of [18F]4-(4-fluorophenyl)-1,2,4-triazole-3,5-dione: an agent for specific radiolabelling of tyrosine

“…[18][19][20] But these reactions are not transposable in [ 18 F] radiochemistry due to the long synthesis time (20-24 h). Recently, Ban and co-workers have reported a tyrosine bioconjugation through ene-type reactions 21,22 (Scheme 1). In this reaction, the cyclic diazodicarboxamide PTAD, an electrophilic compound, reacts selectively on the o-position of the phenol side chain of tyrosine in mild aqueous conditions (aqueous buffer).…”