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.     

A Genetically Encoded Isonitrile Lysine for Orthogonal Bioorthogonal Labeling Schemes

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.     

Metal‐Free Synthetic Approach to 3‐Monosubstituted Unsymmetrical 1,2,4,5‐Tetrazines Useful for Bioorthogonal Reactions

A facile, efficient and metal‐free synthetic approach to 3‐monosubstituted unsymmetrical 1,2,4,5‐tetrazines is presented. Dichloromethane (DCM) is for the first time recognized as a novel reagent in the synthetic chemistry of tetrazines. Using this novel approach 11 3‐aryl/alkyl 1,2,4,5‐tetrazines were prepared in excellent yields (up to 75 %). The mechanism of this new reaction, including the role of DCM in the tetrazine ring formation, has been investigated by ¹³C labeling of DCM, and is also presented and discussed as well as the photophysical and electrochemical properties.     

Bridge-Clamp Bis(tetrazine)s with [N]8 π-Stacking Interactions and Azido-s-Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines

Click chemistry at a tetrazine core is useful for bioorthogonal labeling and crosslinking. Introduced here are two new classes of doubly clickable s-aryl tetrazines synthesized by Cu-catalyzed cross-coupling. Homocoupling of o-brominated s-aryl tetrazines leads to bis(tetrazine)s structurally characterized by tetrazine cores arranged face-to-face. [N]₈ π-stacking interactions are essential to the conformation. Upon inverse electron demand Diels–Alder (iEDDA) cycloaddition, the bis(tetrazine)s produce a unique staple structure. The o-azidation of s-aryl tetrazines introduces a second proximal intermolecular clickable function that leads to double click chemistry opportunities. The stepwise introduction of fluorophores and then iEDDA cycloaddition, including bioconjugation to antibodies, was achieved on this class of tetrazines. This method extends to (thio)etherification, phosphination, trifluoromethylation and the introduction of various bioactive nitrogen-based heterocycles.     

Vinylboronic Acids as Fast Reacting,Synthetically Accessible,and Stable Bioorthogonal Reactants in the Carboni–Lindsey Reaction

Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non‐strained, synthetically accessible and water‐soluble reaction partners in a bioorthogonal inverse electron‐demand Diels–Alder (iEDDA) reaction with 3,6‐dipyridyl‐s‐tetrazines is described. Depending on the substituents, VBA derivatives give second‐order rate constants up to 27 m ^?1 s^?1 in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non‐toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain‐promoted alkyne–azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox.     

Bridge‐Clamp Bis(tetrazine)s with [N]8 π‐Stacking Interactions and Azido‐s‐Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines

Click chemistry at a tetrazine core is useful for bioorthogonal labeling and crosslinking. Introduced here are two new classes of doubly clickable s‐aryl tetrazines synthesized by Cu‐catalyzed cross‐coupling. Homocoupling of o‐brominated s‐aryl tetrazines leads to bis(tetrazine)s structurally characterized by tetrazine cores arranged face‐to‐face. [N]₈ π‐stacking interactions are essential to the conformation. Upon inverse electron demand Diels–Alder (iEDDA) cycloaddition, the bis(tetrazine)s produce a unique staple structure. The o‐azidation of s‐aryl tetrazines introduces a second proximal intermolecular clickable function that leads to double click chemistry opportunities. The stepwise introduction of fluorophores and then iEDDA cycloaddition, including bioconjugation to antibodies, was achieved on this class of tetrazines. This method extends to (thio)etherification, phosphination, trifluoromethylation and the introduction of various bioactive nitrogen‐based heterocycles.     

Synthesis of i-Corona[6]arenes for Selective Anion Binding: Interdependent and Synergistic Anion–π and Hydrogen-Bond Interactions

i-Corona[3]arene[3]tetrazines were synthesized from the nucleophilic aromatic substitution reaction of resorcinol and its derivatives with 3,6-dichlorotetrazine in a one-pot fashion under mild conditions. All of the resulting macrocycles adopted 1,3,5-alternate conformation irrespective of the nature of the substituents on both upper- and lower-rims. i-Corona[3]arene[3]tetrazine was found to self-regulate its macrocyclic conformation and cavity to recognize anions with binding constants spanning from 26 M⁻¹ to 2.2×10³ M⁻¹ depending on the structure of the anions. The selective binding resulted from a significant interdependent and synergistic effect between multiple tetrazine π/anion and C_aryl–H/anion hydrogen bond interactions. Taking advantage of synergistic effect revealed, a cyanobenzene-embedded i-corona[3]arene[3]tetrazine was designedly synthesized and highly selective and very strong affinity toward nitrate with a binding constant of 2.2×10⁵ M⁻¹ was achieved.     

Synthesis and Reactivity Comparisons of 1‐Methyl‐3‐Substituted Cyclopropene Mini‐tags for Tetrazine Bioorthogonal Reactions

Substituted cyclopropenes have recently attracted attention as stable “mini‐tags” that are highly reactive dienophiles with the bioorthogonal tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1‐methyl‐3‐substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels–Alder cycloadditions with 1,2,4,5‐tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3‐amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1‐methyl‐3‐substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated tetrazine ligations. The effect of tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3‐amidomethyl substituted methylcyclopropene reacts faster than trans‐cyclooctenol with a sterically hindered and extremely stable tert‐butyl substituted tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.     

Alkene–Tetrazine Ligation for Imaging Cellular DNA

5‐Vinyl‐2′‐deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels–Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5‐ethynyl‐2′‐deoxyuridine (EdU). The VdU–tetrazine ligation reaction is rapid (k≈0.02 M ^?1 s^?1) and chemically orthogonal to the alkyne–azide “click” reaction of EdU‐modified DNA. Alkene–tetrazine ligation reactions provide the first alternative to azide–alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time‐resolved, multicolor labeling of DNA synthesis.     

Vinylboronic Acids as Fast Reacting,Synthetically Accessible,and Stable Bioorthogonal Reactants in the Carboni–Lindsey Reaction

Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non‐strained, synthetically accessible and water‐soluble reaction partners in a bioorthogonal inverse electron‐demand Diels–Alder (iEDDA) reaction with 3,6‐dipyridyl‐s‐tetrazines is described. Depending on the substituents, VBA derivatives give second‐order rate constants up to 27 m ⁻¹ s⁻¹ in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non‐toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain‐promoted alkyne–azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox.     

Synthesis and Electrochemical Behavior of Electron‐Rich s‐Tetrazine and Triazolo‐tetrazine Nitrate Esters

We have prepared energetic nitrate ester derivatives of 1,2,4,5‐tetrazine and 1,2,4‐triazolo[4,3‐b]‐[1,2,4,5]‐tetrazine ring systems as model compounds to study the electrochemical behavior of tetrazines in the presence of explosive groups. The model compounds showed lower thermal stabilities relative to PETN (pentaerythritol tetranitrate), but slightly improved mechanical sensitivities. The presence of electron‐rich amine donors leads to a cathodic shift of the tetrazine redox potentials relative to those of previously reported tetrazine explosives. At these potentials, electron‐rich tetrazines with either covalently bound or co‐dissolved nitrate ester groups are irreversibly reduced. Effectively, changes in the electronic structure of tetrazines affect their electrochemical response to the presence of nitrate ester groups. Thus, it may be possible to develop tetrazine‐based electrochemical sensors for the detection of specific explosives and electrocatalysts for their disposal.     

In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live‐Cell Imaging Probes

In spite of the wide application potential of 1,2,4,5‐tetrazines, particularly in live‐cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)‐3‐substituted 6‐alkenyl‐1,2,4,5‐tetrazine derivatives through an elimination–Heck cascade reaction. By using this strategy, we provide 24 examples of π‐conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta‐1,3‐diene‐substituted tetrazines, and a diverse array of fluorescent probes suitable for live‐cell imaging. These highly conjugated probes show very strong fluorescence turn‐on (up to 400‐fold) when reacted with dienophiles such as cyclopropenes and trans‐cyclooctenes, and we demonstrate their application for live‐cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live‐cell imaging.     

A Minimal,Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

The unstrained S‐allyl cysteine amino acid was site‐specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5‐tetrazines by means of an inverse‐electron‐demand Diels–Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre‐targeting approach. The small size, easy chemical installation, and selective reactivity of the S‐allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.     

Direct Cu-mediated aromatic 18F-labeling of highly reactive tetrazines for pretargeted bioorthogonal PET imaging

Pretargeted imaging can be used to visualize and quantify slow-accumulating targeting vectors with short-lived radionuclides such as fluorine-18 – the most popular clinically applied Positron Emission Tomography (PET) radionuclide. Pretargeting results in higher target-to-background ratios compared to conventional imaging approaches using long-lived radionuclides. Currently, the tetrazine ligation is the most popular bioorthogonal reaction for pretargeted imaging, but a direct ¹⁸F-labeling strategy for highly reactive tetrazines, which would be highly beneficial if not essential for clinical translation, has thus far not been reported. In this work, a simple, scalable and reliable direct ¹⁸F-labeling procedure has been developed. We initially studied the applicability of different leaving groups and labeling methods to develop this procedure. The copper-mediated ¹⁸F-labeling exploiting stannane precursors showed the most promising results. This approach was then successfully applied to a set of tetrazines, including highly reactive H-tetrazines, suitable for pretargeted PET imaging. The labeling succeeded in radiochemical yields (RCYs) of up to approx. 25%. The new procedure was then applied to develop a pretargeting tetrazine-based imaging agent. The tracer was synthesized in a satisfactory RCY of ca. 10%, with a molar activity of 134 ± 22 GBq μmol⁻¹ and a radiochemical purity of >99%. Further evaluation showed that the tracer displayed favorable characteristics (target-to-background ratios and clearance) that may qualify it for future clinical translation.

A simple, scalable and reliable direct ¹⁸F-labeling procedure has been developed and applied to obtain a pretargeting tetrazine-based imaging agent with favorable in vivo characteristics.     

A Minimal,Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

The unstrained S‐allyl cysteine amino acid was site‐specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5‐tetrazines by means of an inverse‐electron‐demand Diels–Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre‐targeting approach. The small size, easy chemical installation, and selective reactivity of the S‐allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.     

Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.     

Practical synthesis of bis-substituted tetrazines with two pendant 2-pyrrolyl or 2-thienyl groups, precursors of new conjugated polymers

Novel linear oligoheterocycles based on substituted tetrazines are described. The desired compounds have been accomplished by a variation of the original Pinner [Ann. Chem., 297 (1897) 221] synthesis in which the aromatic nitrile reacted with hydrazine in an aqueous solution to give bis(pyrrolyl)tetrazines or bis(phenyl)tetrazines. The bis(phenyl)tetrazines reacted with 3,4-ethylenedioxy-2-(trimethyltin)thiophene or 2-(trimethyltin)thiophene in the presence of Pd(PPh₃)₂Cl₂ or Pd(PPh₃)₄ as catalyst to give the desired compounds. Quantum-chemical calculations were performed to assess the usefulness of the synthesized compounds for electropolymerization. Studies have indicated qualitative difference between bis-pyrrole tetrazine and bis-phenyl tetrazines regarding the electronic density rearrangement due to the loss of an electron.     

Acyclische isomere von aminofluorsilanen,intramolekulare cyclisierung

Fluorosilanes react with lithiated amines to yield aminofluorosilanes. Iso- and sec-butylaminofluorosilanes with bulky ligands form the isomers (I–VII). The cyclisation of monoorganofluorosilanes depends on the size of the substituents. IV and V dimerise by HF-elimination (VIII, IX) and VII cyclises by migration of a methanide ion (X). The mass, ¹H, ¹⁹F and ²⁹Si NMR spectra of the compounds are reported.     

Studies on the stability and stabilization of trans-cyclooctenes through radical inhibition and silver (I) metal complexation

Conformationally strained trans-cyclooctenes (TCOs) engage in bioorthogonal reactions with tetrazines with second order rate constants that can exceed 10⁶ M^?1s^?1. The goal of this study was to provide insight into the stability of TCO reagents and to develop methods for stabilizing TCO reagents for long-term storage. The radical inhibitor Trolox suppresses TCO isomerization under high thiol concentrations and TCO shelf-life can be greatly extended by protecting them as stable Ag(I) metal complexes. ¹H NMR studies show that Ag-complexation is thermodynamically favorable but the kinetics of dissociation are very rapid, and TCO?AgNO₃ complexes are immediately dissociated upon addition of NaCl which is present in high concentration in cell media. The AgNO₃ complex of a highly reactive s-TCO-TAMRA conjugate was shown to label a protein-tetrazine conjugate in live cells with faster kinetics and similar labeling yield relative to a ‘traditional’ TCO-TAMRA conjugate.     

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

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.