Divergent Synthesis of Monosubstituted and Unsymmetrical 3,6‐Disubstituted Tetrazines from Carboxylic Ester Precursors

Abstract

Since tetrazines are important tools to the field of bioorthogonal chemistry, there is a need for new approaches to synthesize unsymmetrical and 3‐monosubstituted tetrazines. Described here is a general, one‐pot method for converting (3‐methyloxetan‐3‐yl)methyl carboxylic esters into 3‐thiomethyltetrazines. These versatile intermediates were applied to the synthesis of unsymmetrical tetrazines through Pd‐catalyzed cross‐coupling and in the first catalytic thioether reduction to access monosubstituted tetrazines. This method enables the development of new tetrazine compounds possessing a favorable combination of kinetics, small size, and hydrophilicity. It was applied to a broad range of aliphatic and aromatic ester precursors and to the synthesis of heterocycles including BODIPY fluorophores and biotin. In addition, a series of tetrazine probes for monoacylglycerol lipase (MAGL) were synthesized and the most reactive one was applied to the labeling of endogenous MAGL in live cells.

Uncovering the key role of distortion in tetrazine ligations guides the design of bioorthogonal tools that defy the reactivity/stability tradeoff

“…Very recently, Joseph Fox and coworkers have reported on the increased IEDDA reactivity of vinyl ether-substituted Tz. [29] Inspired by these results, we hypothesized that the observed unexpected boost in reactivity is due to lowered distortion energies, caused by intramolecular O-N-repulsion (Fig. 6a).…”

“…These applications have motivated and fueled the development of advanced procedures for the synthesis of tetrazine scaffolds. [27][28][29][30][31] In particular, 2-pyridyl-Tz are frequently used despite limited stability, because of their exceptionally high reactivity. This is commonly attributed to the electronwithdrawing effect of the 2-pyridyl substituent, resulting in a lowered orbital energy of the tetrazine, and thereby accelerating the IEDDA cycloaddition.…”

Rapid, traceless and facile peptide cyclization enabled by tetrazine‐thiol exchange

“…Synthesis of oxetane ester 1 was adapted from a protocol described in literature. 27 In a dry round-bottom flask 3-methyl-3-oxetanemethanol (0.630 mL, 6.31 mmol, 1.1 equiv. ), EDCÁHCl (1.07 g, 6.89 mmol, 1.2 equiv.)…”

“…Compound 2 was synthesized following a protocol described in literature. 27 In brief, thiocarbohydrazide (2.00 g, 18.8 mmol, 1.0 equiv. ), methyl iodide (1.29 mL, 20.7 mmol, 1.1 equiv.)…”

Solid-Phase Synthesis of s-Tetrazines

“…These formed thioimidate esters in situ with subsequent nucleophilic attack by hydrazine leading to regeneration of the thiol and formation of an amidrazone, which then reacted with another equivalent of thioimidate ester to give, after oxidation, the tetrazine ( Scheme 1 ). Recently, Fox 33 developed a one-pot method for the synthesis of 3-thiomethyltetrazines from carboxylic esters, with the 3-thiomethyltetrazines used in thioether reduction or palladium-catalyzed cross-coupling chemistries to generate mono- and disubstituted aliphatic or aromatic tetrazines (60–80% yield). Here, we report an expedient solid-phase synthesis route to both monosubstituted and unsymmetrical disubstituted s -tetrazines, bearing different functional groups, based on the thiol-promoted reaction between supported aryl nitriles and hydrazine.…”