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.     

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.     

Long‐Term Live‐Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI‐SiR

Super‐resolution imaging of live cells over extended time periods with high temporal resolution requires high‐density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high‐density plasma membrane probe DiI‐TCO and the photostable STED dye SiR‐Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI‐SiR. Using DiI‐SiR, we visualized filopodia dynamics in HeLa cells over 25 min at 0.5 s temporal resolution, and visualized dynamic contact‐mediated repulsion events in primary mouse hippocampal neurons over 9 min at 2 s temporal resolution. HIDE probes such as DiI‐SiR are non‐toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super‐resolution over biologically relevant timescales.     

Mechanism‐Based Fluorogenic trans‐Cyclooctene–Tetrazine Cycloaddition

The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans ‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Targeted Imaging and Proteomic Analysis of Tumor‐Associated Glycans in Living Animals

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor‐associated glycans by using ligand‐targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer‐cell‐specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue‐specific manner. Subsequently, a copper‐free click reaction was performed in vivo to chemoselectively conjugate the azides with a near‐infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.     

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.     

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.     

Stable Pyrrole‐Linked Bioconjugates through Tetrazine‐Triggered Azanorbornadiene Fragmentation

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.     

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.     

Origin of Orthogonality of Strain‐Promoted Click Reactions

Site‐specific labeling of biomolecules is rapidly advancing due to the discovery of novel mutually orthogonal reactions. Quantum chemistry studies have also increased our understanding of their relative rates, although these have until now been based on highly simplified reactants. Here we examine a set of strain‐promoted click‐type cycloaddition reactions of n‐propyl azide, 3‐benzyl tetrazine and 3‐benzyl‐6‐methyl tetrazine with cyclooctenes/ynes, in which we aim to address all relevant structural details of the reactants. Our calculations have included the obligatory handles used to attach the label and biomolecule as these can critically influence the stereochemistry and electron demand of the reaction. We systematically computed orbital gaps, activation and distortion energies using density functional theory and determined experimental rates for validation. Our results challenge the current paradigm of the inverse electron demand for this class of reactions. We found that the ubiquitous handles, when next to the triple bond of cyclooctynes, can switch the Diels–Alder type ligations to normal electron demand, a class we term as SPINEDAC reactions. Electron donating substituents on tetrazine can enhance normal demand but also increase distortion penalties. The presence and isomeric configuration of handles thus determine the reaction speed and regioselectivity. Our findings can be directly utilized in engineering genuine cycloaddition click chemistries for biological labeling.     

Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra‐fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans‐cyclooctene (TCO)‐mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t_1/2 <1 s) than predicted. This allowed multi‐cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.     

Cross‐Platform DNA Encoding for Single‐Cell Imaging of Gene Expression

Integration of imaging data across different molecular target types can provide in‐depth insight into cell physiology and pathology, but remains challenging owing to poor compatibility between target‐type‐specific labeling methods. We show that cross‐platform imaging analysis can be readily achieved through DNA encoding of molecular targets, which translates the molecular identity of various target types into a uniform in situ array of ssDNA tags for subsequent labeling with complementary imaging probes. The concept was demonstrated through multiplexed imaging of mRNAs and their corresponding proteins with multicolor quantum dots. The results reveal heterogeneity of cell transfection with siRNA and outline disparity in RNA interference (RNAi) kinetics at the level of both the mRNA and the encoded protein.