Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click-to-Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse-electron-demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.     

Pre‐targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles

The pre‐targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions—the condensation reaction of aromatic nitriles and aminothiols and the inverse‐electron demand Diels–Alder reaction between tetrazine and trans‐cyclooctene (TCO)—to develop a novel strategy for pre‐targeted imaging of the activity of proteases. The substrate probe ( TCO‐C‐SNAT4 ) can be selectively activated by an enzyme target (e.g. caspase‐3/7), which triggers macrocyclization and subsequent in situ self‐assembly into nanoaggregates retained at the target site. The tetrazine‐imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO‐C‐SNAT4 can be repeatedly injected to generate and accumulate more TCO‐nanoaggregates for click labeling.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

Bioorthogonal Turn‐On Probe Based on Aggregation‐Induced Emission Characteristics for Cancer Cell Imaging and Ablation

Bioorthogonal turn‐on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn‐on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn‐on probes based on red‐emissive fluorogens with aggregation‐induced emission characteristics (AIEgens). The probe is water soluble and non‐fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide‐functionalized glycans on cancer cell surface. The fluorescence turn‐on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.     

Development of a 18F‐Labeled Tetrazine with Favorable Pharmacokinetics for Bioorthogonal PET Imaging

A low‐molecular‐weight ¹⁸F‐labeled tetrazine derivative was developed as a highly versatile tool for bioorthogonal PET imaging. Prosthetic groups and undesired carrying of ¹⁸F through additional steps were evaded by direct ¹⁸F‐fluorination of an appropriate tetrazine precursor. Reaction kinetics of the cycloaddition with trans‐cyclooctenes were investigated by applying quantum chemical calculations and stopped‐flow measurements in human plasma; the results indicated that the labeled tetrazine is suitable as a bioorthogonal probe for the imaging of dienophile‐tagged (bio)molecules. In vitro and in vivo investigations revealed high stability and PET/MRI in mice showed fast homogeneous biodistribution of the ¹⁸F‐labeled tetrazine that also passes the blood–brain barrier. An in vivo click experiment confirmed the bioorthogonal behavior of this novel tetrazine probe. Due to favorable chemical and pharmacokinetic properties this bioorthogonal agent should find application in bioimaging and biomedical research.     

Pre-targeted Imaging of Protease Activity through In Situ Assembly of Nanoparticles

The pre-targeted imaging of enzyme activity has not been reported, likely owing to the lack of a mechanism to retain the injected substrate in the first step for subsequent labeling. Herein, we report the use of two bioorthogonal reactions—the condensation reaction of aromatic nitriles and aminothiols and the inverse-electron demand Diels–Alder reaction between tetrazine and trans-cyclooctene (TCO)—to develop a novel strategy for pre-targeted imaging of the activity of proteases. The substrate probe ( TCO-C-SNAT4 ) can be selectively activated by an enzyme target (e.g. caspase-3/7), which triggers macrocyclization and subsequent in situ self-assembly into nanoaggregates retained at the target site. The tetrazine-imaging tag conjugate labels TCO in the nanoaggregates to generate selective signal retention for imaging in vitro, in cells, and in mice. Owing to the decoupling of enzyme activation and imaging tag immobilization, TCO-C-SNAT4 can be repeatedly injected to generate and accumulate more TCO-nanoaggregates for click labeling.     

Ultrafluorogenic Monochromophore-Type BODIPY-Tetrazine Series for Dual-Color Bioorthogonal Imaging with a Single Probe

Various fluorogenic probes utilizing tetrazine (Tz) as a fluorescence quencher and bioorthogonal reaction partner have been extensively studied over the past few decades. Herein, we synthesized a series of boron-dipyrromethene (BODIPY)-Tz probes using monochromophoric design strategy for bioorthogonal cellular imaging. The BODIPY-Tz probes exhibited excellent bicyclo[6.1.0]nonyne (BCN)-selective fluorogenicity with three- to four-digit-fold enhancements in fluorescence over a wide range of emission wavelengths, including the far-red region. Furthermore, we demonstrated the applicability of BODIPY-Tz probes in bioorthogonal fluorescence imaging of cellular organelles without washing steps. We also elucidated the aromatized pyridazine moiety as the origin of BCN-selective fluorogenic behavior. Additionally, we discovered that the fluorescence of the trans-cyclooctene (TCO) adducts was quenched in aqueous media via photoinduced electron transfer (PeT) process. Interestingly, we observed a distinctive recovery of the initially quenched fluorescence of BODIPY-Tz-TCO upon exposure to hydrophobic media, accompanied by a significant bathochromic shift of its emission wavelength relative to that exhibited by the corresponding BODIPY-Tz-BCN. Leveraging this finding, for the first time, we achieved dual-color bioorthogonal cellular imaging with a single BODIPY-Tz probe.     

Click‐to‐Release from trans‐Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage

The bioorthogonal cleavage of allylic carbamates from trans‐cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4‐dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p‐aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO‐masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine‐free medium.     

New Red‐Emitting Tetrazine‐Phenoxazine Fluorogenic Labels for Live‐Cell Intracellular Bioorthogonal Labeling Schemes

The synthesis of a set of tetrazine‐bearing fluorogenic dyes suitable for intracellular labeling of proteins in live cells is presented. The red excitability and emission properties ensure minimal autofluorescence, while through‐bond energy‐transfer‐based fluorogenicity reduces nonspecific background fluorescence of unreacted dyes. The tetrazine motif efficiently quenches fluorescence of the phenoxazine core, which can be selectively turned on chemically upon bioorthogonal inverse‐electron‐demand Diels–Alder reaction with proteins modified genetically with strained trans‐cyclooctenes.     

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.     

Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells

The inverse‐electron‐demand Diels–Alder (iDA) reaction has recently been repurposed as a bioorthogonal decaging reaction by accelerating the elimination process after an initial cycloaddition between trans‐cyclooctene (TCO) and tetrazine (TZ). Herein, we systematically surveyed 3,6‐substituted TZ derivatives by using a fluorogenic TCO–coumarin reporter followed by LC‐MS analysis, which revealed that the initial iDA cycloaddition step was greatly accelerated by electron‐withdrawing groups (EWGs) while the subsequent elimination step was strongly suppressed by EWGs. In addition, smaller substituents facilitated the decaging process. These findings promoted us to design and test unsymmetric TZs bearing an EWG group and a small non‐EWG group at the 3‐ and 6‐position, respectively. These TZs showed remarkably enhanced decaging rates, enabling rapid iDA‐mediated protein activation in living cells.     

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.     

Super‐Resolution Imaging of the Golgi in Live Cells with a Bioorthogonal Ceramide Probe

We report a lipid‐based strategy to visualize Golgi structure and dynamics at super‐resolution in live cells. The method is based on two novel reagents: a trans‐cyclooctene‐containing ceramide lipid (Cer‐TCO) and a highly reactive, tetrazine‐tagged near‐IR dye (SiR‐Tz). These reagents assemble via an extremely rapid “tetrazine‐click” reaction into Cer‐SiR, a highly photostable “vital dye” that enables prolonged live‐cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer‐SiR is nontoxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi‐resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.     

A General Strategy for Macrotheranostic Prodrug Activation: Synergy between the Acidic Tumor Microenvironment and Bioorthogonal Chemistry

Prodrugs activated by endogenous stimuli face the problem of tumor heterogeneity. Bioorthogonal prodrug activation that utilizes an exogenous click reaction has the potential to solve this problem, but most of the strategies currently used rely on the presence of endogenous receptors or overexpressed enzymes. We herein integrate the acidic, extracellular microenvironment of a tumor and a click reaction as a general strategy for prodrug activation. This was achieved by using a tumor pH‐responsive polymer containing tetrazine groups, which formed unreactive micelles in the blood but disassembled in response to tumor pH. The vinyl ether group on the macrotheranostic prodrug (CyPVE) is activated by the tetrazine groups, which was confirmed by tumor‐specific fluorescence activation and phototoxicity restoration. Therefore, the bioorthogonal reactions in the context of the ubiquitous acidic tumor microenvironment can provide a general strategy for bioorthogonal prodrug activation.     

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.     

Conditional Singlet Oxygen Generation through a Bioorthogonal DNA‐targeted Tetrazine Reaction

We report the use of bioorthogonal reactions as an original strategy in photodynamic therapy to achieve conditional phototoxicity and specific subcellular localization simultaneously. Our novel halogenated BODIPY‐tetrazine probes only become efficient photosensitizers (Φ_Δ≈0.50) through an intracellular inverse‐electron‐demand Diels–Alder reaction with a suitable dienophile. Ab initio computations reveal an activation‐dependent change in decay channels that controls ¹O₂ generation. Our bioorthogonal approach also enables spatial control. As a proof‐of‐concept, we demonstrate the feasibility of the selective activation of our dormant photosensitizer in cellular nuclei, causing cancer cell death upon irradiation. Thus, our dual biorthogonal, activatable photosensitizers open new venues to combat current limitations of photodynamic therapy.     

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.     

A Systematic Study of Coumarin–Tetrazine Light-Up Probes for Bioorthogonal Fluorescence Imaging

Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin–tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.     

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications

The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.     

Strain‐Promoted Reaction of 1,2,4‐Triazines with Bicyclononynes

Strain‐promoted inverse electron‐demand Diels–Alder cycloaddition (SPIEDAC) reactions between 1,2,4,5‐tetrazines and strained dienophiles, such as bicyclononynes, are among the fastest bioorthogonal reactions. However, the synthesis of 1,2,4,5‐tetrazines is complex and can involve volatile reagents. 1,2,4‐Triazines also undergo cycloaddition reactions with acyclic and unstrained dienophiles at elevated temperatures, but their reaction with strained alkynes has not been described. We postulated that 1,2,4‐triazines would react with strained alkynes at low temperatures and therefore provide an alternative to the tetrazine cycloaddition reaction for use in in vitro or in vivo labelling experiments. We describe the synthesis of a 1,2,4‐triazin‐3‐ylalanine derivative fully compatible with the fluorenylmethyloxycarbonyl (Fmoc) strategy for peptide synthesis and demonstrate its reaction with strained bicyclononynes at 37 °C with rates comparable to the reaction of azides with the same substrates. The synthetic route to triazinylalanine is readily adaptable to late‐stage functionalization of other probe molecules, and the 1,2,4‐triazine‐SPIEDAC therefore has potential as an alternative to tetrazine cycloaddition for applications in cellular and biochemical studies.