Mechanisms and Substituent Effects of Metal-Free Bioorthogonal Reactions.

Reactions that occur under physiological conditions find diverse uses in the chemical and biological sciences. However, the limitations that biological systems place on chemical reactions restrict the number of such bioorthogonal reactions. A profound understanding of the mechanistic principles and structure-reactivity trends of these transformations is therefore critical to access new and improved versions of bioorthogonal chemistry. The present article reviews the mechanisms and substituent effects of some of the principal metal-free bioorthogonal reactions based on inverse-electron demand Diels-Alder reactions, 1,3-dipolar cycloadditions, and the Staudinger reaction. Mechanisms of modified versions that link these reactions to a dissociative step are further discussed. The presented summary is anticipated to aid the advancement of bioorthogonal chemistry.

Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups.

In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions.

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

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 ß-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.

Stable, Reactive, and Orthogonal Tetrazines: Dispersion Forces Promote the Cycloaddition with Isonitriles.

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure-activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol-1 s-1 were accessible by combining bulky and electron-withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles.

Dissociative Bioorthogonal Reactions.

Bioorthogonal reactions that proceed readily under physiological conditions without interference from biomolecules have found widespread application in the life sciences. Complementary to the bioorthogonal reactions that ligate two molecules, reactions that release a molecule or cleave a linker are increasingly attracting interest. Such dissociative bioorthogonal reactions have a broad spectrum of uses, for example, in controlling bio-macromolecule activity, in drug delivery, and in diagnostic assays. This review article summarizes the developed bioorthogonal reactions linked to a release step, outlines representative areas of the applications of such reactions, and discusses aspects that require further improvement.

Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables the Controlled Release of Fluorophores and Drugs in Vivo.

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopropyl derivatives react with tetrazines to afford 3-oxopropyl groups that eliminate diverse functionalities. The study shows that the reaction is rapid and can liberate phenols and amines near-quantitatively under physiological conditions. The reaction is compatible with living organisms as demonstrated by the release of a resorufin fluorophore and a mexiletine drug in zebrafish embryos implanted with tetrazine-modified beads. The combined benefits of synthetic ease, rapid kinetics, diversity of leaving groups, high release yields, and structural compactness, make 3-isocyanopropyl derivatives attractive chemical caging moieties for uses in chemical biology and drug delivery.

Rapid and efficient tetrazine-induced drug release from highly stable benzonorbornadiene derivatives.

A novel class of bioorthogonal release reactions based on benzonorbornadiene derivatives was developed. These carrier molecules are highly stable at physiological conditions, but react rapidly with 1,2,4,5-tetrazines, and near-quantitatively release cargo molecules such as drugs and optical reporters.