Tuning Isonitrile/Tetrazine Chemistry for Accelerated Deprotection and Formation of Stable Conjugates

Abstract

The isocyano group is a valuable functionality for bioorthogonal reactions because it rapidly reacts with tetrazines to either form stable conjugates or release payloads from 3-isocyanopropyl groups. Here we provide mechanistic insights into the dissociative steps that follow the initial cycloaddition and analyze how structural modifications affect these processes. Three main outcomes of this study have important implications for designing such groups for bioorthogonal applications. First, anion-stabilizing substituents at C-2 of the 3-isocyanopropyl group promote β-elimination and accelerate deprotection. Second, tetrazines with bulky substituents form stable imine conjugates even with primary isonitriles that are otherwise rapidly hydrolyzed. Third, the elimination step is independent from hydrolysis to the aldehyde and instead can occur directly from the imine intermediate. These findings will allow tuning the structures of tetrazine and isonitrile reactants for application in bioorthogonal ligation and release chemistry.

The Unique Bioorthogonal Chemistry of Isonitriles

“…However, we recently showed that even simple primary isonitriles can form stable linkages with tetrazines that have bulky substituents. 36 The adduct of 3,6bis-tert-butyl-1,2,4,5-tetrazine and n-butyl isocyanide was stable in D 2 O/DMSO-d 6 (1:1), with a gradual hydrolysis of 18% in 72 hours. 36 Based on this result, it is now possible to exploit the structural compactness of the isocyano group for labeling applications, opening new opportunities in chemical biology.…”

“…36 The adduct of 3,6bis-tert-butyl-1,2,4,5-tetrazine and n-butyl isocyanide was stable in D 2 O/DMSO-d 6 (1:1), with a gradual hydrolysis of 18% in 72 hours. 36 Based on this result, it is now possible to exploit the structural compactness of the isocyano group for labeling applications, opening new opportunities in chemical biology. In another bioorthogonal reaction, simple isonitriles react with chlorooximes under physiological conditions forming -hydroximino amide linkages (Scheme 2).…”

Isocyanides: Promising Functionalities in Bioorthogonal Labeling of Biomolecules

“…Meanwhile, Franzini’s group thoroughly explored the structure-activity relationship of the reaction between isocyanides and tetrazines ( Table 1 ), and found that tetrazines with bulky substituents could form stable adducts with primary isocyanides, thus obviating the need for engineering the isocyanide moiety (e.g., tertiary isocyanides) ( Tu et al, 2019 ; Xu et al, 2019 ). What’s more interesting is that stable asymmetric tetrazines with bulky and electron-withdrawing substituents ( Tz-4 ) can react with isocyanides rapidly under high-water conditions ( Table 1 , Entries 12–13).…”

Activation and Delivery of Tetrazine-Responsive Bioorthogonal Prodrugs

“…However, toxic acrolein as a by-product, may impose restrictions on in vivo applications. Further mechanistic study suggests the elimination reaction is hydrolysis-independent and may avoid the generation of acrolein [93].…”