Tetrazines

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Tetrazine represents a crucial group of nitrogen-containing heterocyclic compounds that finds extensive application across chemistry, materials science, and biomedical research. Tetrazine compounds exhibit both high reactivity and stability because of their unique molecular structure, which makes them perfect candidates for synthetic chemistry applications as well as fluorescence labeling and drug development, including antibody-drug conjugates. BOC Sciences provides multiple tetrazine compound options that cover tetrazine dyes and derivatives along with click chemistry reagents to assist both researchers and industrial clients in their chemical synthesis projects and drug development activities.

What is Tetrazine?

Tetrazine is a heterocyclic molecule comprising a six-membered ring containing four nitrogen atoms. It is extensively utilized in bioorthogonal chemistry, particularly in inverse electron-demand Diels-Alder (IEDDA) reactions, owing to its capacity for rapid and selective reactions with strained alkenes or alkynes, such as trans-cyclooctenes (TCOs). Tetrazine-based reactions have gained prominence for applications necessitating extremely efficient and specialized chemical reactions, especially within biological systems. The following are some representative tetrazine compounds:

Fig. 1. Tetrazine structure (BOC Sciences Authorized).Fig. 1. Tetrazine structure (BOC Sciences Authorized).

  • Tetrazine Acid

Tetrazine acid is a chemical compound with a tetrazine ring structure that contains a carboxyl group (-COOH). It is commonly used as a crosslinking agent or labeling molecule in synthetic chemistry and can react with a variety of chemical groups. Due to its good stability and high reactivity, tetrazine acid has broad applications in bio-labeling, molecular probes, and drug delivery systems. The acidic nature of tetrazine acid makes it suitable for coupling reactions with amino, alcohol, and other chemical groups.

  • Tetrazine Alkyne

Tetrazine alkyne is a derivative of tetrazine compounds that contains an alkyne (-C≡C) group. Due to the high chemical reactivity of the alkyne group, tetrazine alkyne can undergo click chemistry reactions with molecules containing double bonds or alkyne groups. It is commonly used in molecular labeling and crosslinking reactions. Tetrazine alkyne is widely applied in biological imaging, drug delivery systems, and highly efficient bio-labeling, particularly in click chemistry reactions that offer high selectivity and low by-product formation.

  • Tetrazine Amine

Tetrazine amine is a derivative of tetrazine compounds containing an amino (-NH2) functional group. Tetrazine amine exhibits high nucleophilicity and can react with compounds containing carboxyl, ester, or other active groups. It is widely used in protein labeling, drug molecule conjugation, molecular probe synthesis, and functionalization processes in materials science. The chemical reactivity of tetrazine amine results in high selectivity in reactions between biological molecules.

  • Tetrazine Biotin

Tetrazine biotin is a conjugate of tetrazine compounds and biotin molecules. Biotin is an important biological labeling molecule that can bind with avidin or streptavidin. Tetrazine biotin combines the high reactivity of the tetrazine group with the specific binding capability of biotin, making it widely used in bio-labeling, cell tracking, and molecular recognition. It plays a significant role in molecular imaging, targeted drug delivery, and the development of biosensors.

  • Tetrazine NHS Ester

Tetrazine NHS ester is a derivative formed by the conjugation of tetrazine compounds with an NHS ester (N-Hydroxysuccinimide Ester) group. NHS esters are commonly used active ester groups that can undergo efficient coupling reactions with amino and amine compounds. Tetrazine NHS ester is widely used for labeling and crosslinking reactions of proteins, antibodies, and other biomolecules. This compound plays an important role in biomedical research, especially in drug development, molecular labeling, and targeted delivery systems.

  • Tetrazine PEG

Tetrazine PEG is a derivative that combines tetrazine groups with polyethylene glycol (PEG). PEG is a widely used polymer compound that enhances the solubility, stability, and biocompatibility of molecules in vivo. Tetrazine PEG is extensively used in the biomedical field, particularly in drug delivery, molecular labeling, cell targeting, and nanomedicine research. The combination of tetrazine groups and PEG makes it an efficient molecular labeling and crosslinking agent, offering significant advantages in improving drug targeting and bioavailability.

Tetrazine Structure

The basic structure of tetrazine compounds is a six-membered ring containing four nitrogen atoms, typically 1,2,4,5-tetrazine. Tetrazine's structure is stable, but the electron distribution between nitrogen atoms allows it to react rapidly under specific conditions. The most famous tetrazine reaction is the click reaction with certain double bonds (e.g., IEDDA), leading to the formation of crosslinked or labeled compounds. The stability of tetrazine depends on the covalent bond strength between nitrogen atoms within the ring and the influence of the external environment. Despite being relatively stable in itself, tetrazine compounds can react intensely with other reagents under certain conditions, especially in click chemistry reactions. Furthermore, tetrazine derivatives can be modified with different chemical groups to facilitate their binding with various biomolecules or probes.

  • Tetrazine Reaction

Tetrazine's reactivity is mainly evident in its reactions with different types of reagents, one of the most typical being the reaction of tetrazine with alkenes such as cyclohexene or cyclooctene. This reaction generally occurs under mild conditions, generating stable bicyclic compounds that are widely used in labeling, crosslinking, and drug delivery systems.

  • Tetrazine Stability

Tetrazine compounds exhibit relatively high stability, especially when their structure is not affected by external chemical environments. Under suitable pH conditions, tetrazine shows good thermal stability. However, due to the special nature of its nitrogen-containing multiple bonds, tetrazine may undergo depolymerization or degradation in high temperatures, strong acids, or strong bases, requiring careful stability control during synthesis and application.

Synthesis of Tetrazine

The synthesis of tetrazines began in the late 19th century, initially achieved by the Pinner synthesis method. This classic method involves two steps: first, reacting an aromatic nitrile with hydrazine to produce dihydrotetrazine, followed by an oxidation reaction. However, this method is mainly limited to the synthesis of aromatic tetrazines, and alkyl tetrazines containing functional groups are difficult to synthesize or yield lower outputs. Tetrazine compounds play a significant role in modern medicine, so simplifying their synthesis has become a key focus of research. The synergistic effect of transition metals can enhance the nucleophilic reaction between nitriles and hydrazine, providing a new approach for synthesizing tetrazines. Studies have shown that by screening different Lewis acid metals (such as divalent nickel and zinc salts), the synthesis of amino oxime intermediates can be catalyzed, ultimately achieving the synthesis of 3,6-dibenzyl-1,2,4,5-tetrazine with a yield of up to 95%. This method differs from the traditional Pinner synthesis. By precisely adjusting the ratio of two starting nitriles and using a 5% catalyst load, asymmetric tetrazines can be synthesized with moderate to good yields. The resulting asymmetric tetrazines can further undergo derivatization with amino, hydroxyl, or carboxyl functional groups. Additionally, using Lewis acid-assisted one-pot synthesis, starting from formyl salts, can significantly improve the yield of monosubstituted tetrazines. This technique has enabled multiple research teams to consistently synthesize tetrazine derivatives with unique structures, advancing intracellular wash-free fluorescence imaging and pre-targeted 18F and 11C positron emission tomography (PET) studies.

Tetrazine Dye

Tetrazine dyes are a class of fluorescent dyes based on the tetrazine molecular structure. Tetrazine fluorophores, known for their excellent optical properties and chemical stability, are widely used in fields such as biological imaging, molecular probes, chemical labeling, and cell tracking. These dyes exhibit unique fluorescent properties across different spectral ranges, making them indispensable tools in biomedical and chemical research. Typically, tetrazine-based active dyes can be used to label cyclic alkenes (such as cyclooctene, trans-cyclooctene) and other reactive alkenes via the Diels-Alder reaction. The trans-Diels-Alder reaction between cyclic alkenes and tetrazine is known to be the fastest bioorthogonal reaction, enabling real-time labeling of proteins. Tetrazine-based active dyes can fluorescently label molecules conjugated with strained cyclic alkenes. Tetrazine can quench fluorescence emitted in the 510-570 nm wavelength range, but when strained cyclic alkenes react with these tetrazines, fluorescence is restored. This phenomenon of fluorescence quenching and regeneration allows for real-time monitoring of protein movement. The reaction between strained cyclic alkenes and tetrazine can be used for protein labeling in various buffer solutions in vitro, as well as in vivo labeling. Currently, the reaction between strained cyclic alkenes and tetrazine has been successfully applied to antibody labeling, protein modification, and super-resolution imaging.

  • BODIPY Tetrazine

BODIPY (Boron-dipyrromethene) series dyes are commonly used in fluorescence labeling due to their high fluorescence efficiency, good stability, and relatively long fluorescence lifetime. By conjugating BODIPY dyes with tetrazine groups, BODIPY-Tetrazine complexes are formed. These dyes are widely used in biological imaging technologies due to their excellent optical properties. BODIPY-Tetrazine exhibits strong light absorption and high quantum yield, providing bright and stable fluorescence signals within cells. This makes BODIPY-Tetrazine an ideal labeling molecule for cell tracking, live imaging, and real-time monitoring of molecular interactions. Additionally, the synthesis route for BODIPY-Tetrazine is relatively straightforward, making it highly applicable in drug delivery systems and targeted therapy.

  • Tetrazine Cy5

Tetrazine Cy5 dye combines the properties of tetrazine and Cy5 (a common near-infrared fluorescent dye), offering extremely high photostability and specific fluorescence emission characteristics within a particular wavelength range. Cy5 dye is already widely used in biological imaging, mainly due to its emission wavelength in the near-infrared region (approximately 660–700 nm), which allows it to penetrate tissues and reduce background signals. By combining the tetrazine group with Cy5 dye, Tetrazine Cy5 enables efficient labeling and tracking of target molecules (such as proteins, nucleic acids, etc.) through chemical reactions. The combination of tetrazine with Cy5 enhances the dye's reactivity and selectivity, making it particularly useful for complex labeling reactions in biological tagging and molecular imaging techniques. This makes Tetrazine Cy5 a highly useful tool in molecular detection, cell analysis, and biological labeling.

Tetrazine Click Chemistry

Tetrazine click chemistry is an efficient, highly selective, and mild chemical reaction, classified as a bioorthogonal reaction. This reaction utilizes tetrazine as an electron-deficient dienophile, reacting with electron-rich diene molecules (such as cyclooctene or norbornene) to form new chemical bonds. This reaction is characterized by its rapid pace and the absence of by-products (such as nitrogen and carbon dioxide), making it widely applicable in biological labeling, drug delivery systems, and molecular probe synthesis. The introduction of click chemistry has greatly expanded the application range of tetrazine compounds, especially in drug development and biological research. Through click chemistry, tetrazine can efficiently couple with other biomolecules (such as antibodies, peptides, DNA, etc.), enabling its use in precise drug delivery, molecular detection, and cell tracking.

In the IEDDA reaction, tetrazine acts as an electron-deficient diene due to the electron-withdrawing nature of its nitrogen atoms. It reacts with electron-rich dienophiles, typically strained alkenes like trans-cyclooctene (TCO), norbornene, or other cyclic alkenes. This reaction occurs rapidly under mild conditions and is one of the fastest bioorthogonal reactions known, often referred to as "click chemistry" due to its high efficiency and simplicity. The tetrazine-alkene/alkyne reaction is bioorthogonal, meaning it does not interfere with natural biochemical processes, making it particularly suitable for in vivo applications, where maintaining the integrity of living systems is crucial.

Tetrazine/trans-cyclooctene (Tz/TCO) click and release reagents have played an important role in recent advances due to their excellent reaction kinetics, the absence of hazardous by-products (N2 and CO2), and their outstanding chemical optimizability (allowing adjustment of reagent properties). Robillard and colleagues' ingenious discovery provided the first clue that 3-OH-functionalized TCO derivatives (release-TCO, rTCO) could be used to induce decarboxyl elimination from rTCO through post-click isomerization. Tetrazine click acts as bioorthogonal scissors, cleaving carbamate substituents from rTCO in this inverse electron-demand Diels-Alder (IEDDA) tetrazine elimination. As long as rTCO-based systems can achieve the necessary robustness and efficiency, this opens up unlimited possibilities for in vivo release applications, such as drug delivery, nanomaterials, prodrug strategies, antibody-drug conjugates, and small-molecule conjugates.

Tetrazine Medication

Tetrazine medication refers to drugs developed using the chemical properties of tetrazine compounds, which are typically applied in targeted therapies and molecular labeling. Due to their unique chemical reactivity, tetrazine compounds can efficiently undergo click chemistry reactions with other chemical groups, enabling precise labeling and targeted delivery of drug molecules. In drug development, tetrazine compounds are used to synthesize biomarker molecules, molecular probes, and targeted drug carriers. By binding to biomolecules or drug carriers, tetrazine compounds can enhance drug targeting, stability, and biocompatibility, thereby improving efficacy and reducing side effects. In drug delivery systems, tetrazine compounds are commonly used to conjugate with targeting molecules, antibodies, or drug molecules to improve drug targeting and bioavailability. For instance, tetrazine-based drugs can be combined with chemotherapy agents or radioactive drugs for targeted tumor treatment or precision therapy for other diseases. Additionally, tetrazine compounds are widely used in antibody-drug conjugates (ADCs) and other advanced drug delivery systems. By binding to drug carriers or targeting molecules, tetrazine compounds facilitate targeted drug release, enhancing therapeutic effects. With the continuous advancement of tetrazine chemistry, tetrazine drugs show great potential in precision medicine, cancer therapy, and targeted treatment for other diseases.

What is Tetrazine Used For?

Tetrazine compounds and their derivatives have broad applications in the biopharmaceutical field, especially in pre-targeting imaging, drug development and delivery systems, and bio-conjugation. The following is a detailed description of these applications.

  • Pre-targeting Imaging

The application of tetrazine compounds in pre-targeting imaging primarily lies in their ability to selectively convert dihydrotetrazine into reactive tetrazine through light activation. This novel approach utilizes photouncaging technology, allowing light-triggered reactions in living cells and facilitating bioorthogonal cycloaddition reactions. Specifically, dihydrotetrazine compounds convert into reactive tetrazine under light conditions, and then undergo a cycloaddition reaction with dienophiles (such as trans-cyclooctene), enabling spatiotemporal control. This process provides high temporal and spatial resolution, allowing precise regulation of biomolecule reactions within cells.

  • Delivery Systems

Tetrazine chemistry is applied in drug development, particularly in pre-targeted drug delivery technologies. By utilizing tetrazine click chemistry, researchers can direct targeting molecules to disease sites, such as tumor tissues. The core of this method involves first positioning tetrazine compounds and dienophiles at target cells or tissues, then introducing a drug-tetrazine conjugate. This drug conjugate is activated only when the tetrazine reacts with the dienophile, enabling targeted drug release. The major advantage of this strategy is the precise control over the drug's activity, significantly reducing side effects. The drug is activated only in the diseased area, minimizing toxicity to normal cells. This targeted release system significantly enhances therapeutic efficacy, especially in cancer therapy, ensuring high potency at cancer cells without damaging normal cells.

  • Biopharmaceutical Field

Tetrazine's applications in the biopharmaceutical field continue to expand. In cancer treatment, tetrazine derivatives are being studied as targeted drugs that can specifically bind to tumor cells and trigger drug release, thereby enhancing anticancer efficacy. Furthermore, tetrazine has potential in immunotherapy, gene therapy, and vaccine development, with researchers exploring its applications in these areas.

  • Bio-conjugation

Tetrazine click chemistry is also crucial in bio-conjugation, particularly in covalent linking between biomolecules such as peptides, proteins, or nucleic acids. Tetrazine click reactions are characterized by rapid response rates and excellent selectivity, allowing precise construction of complex chemical structures without disrupting the natural activity of biomolecules. This high-precision conjugation reaction is of great significance in biopharmaceutical research and can be used for the synthesis of various biomarker and functionalized molecules. For example, through tetrazine click chemistry, researchers can link fluorescently labeled molecules to target biomolecules, enabling real-time monitoring of intracellular molecular dynamics. Moreover, this technology can be used to develop novel biosensors, promoting research into biological processes.

  • Bio-labeling and Probes

Tetrazine derivatives are widely used in the field of bio-labeling, especially in fluorescence probes, radioactive labels, and enzyme-linked immunosorbent assay (ELISA) detection methods. Their chemical structure enables rapid reactions with different molecules, often combining with other functionalized molecules (such as fluorescent dyes or radioactive isotopes) to label and detect biological samples. These labels provide important information about cell behavior, molecular interactions, and biological processes.

  • Chemical Reactions and Catalysis

Tetrazine is also used as a catalyst in chemical reactions. In certain chemical synthesis reactions, tetrazine's reactivity significantly improves reaction efficiency, particularly in processes that require selective control of reactions. For example, it is commonly used in organic synthesis for cyclization reactions, reduction reactions, and other processes that demand high chemical activity. By modifying the structure of tetrazine, it is possible to effectively control the catalytic activity and selectivity of reactions.

References:

  1. García-Vázquez R., et al., Recent advances in the development of tetrazine ligation tools for pretargeted nuclear imaging. Pharmaceuticals. 2022, 15(6): 685.
  2. Wu H., et al., Advances in tetrazine bioorthogonal chemistry driven by the synthesis of novel tetrazines and dienophiles. Accounts of chemical research. 2018, 51(5): 1249-1259.
  3. Liu L., et al., Light-activated tetrazines enable precision live-cell bioorthogonal chemistry. Nature chemistry. 2022, 14(9): 1078-1085.
  4. He X., et al., An all-in-one tetrazine reagent for cysteine-selective labeling and bioorthogonal activable prodrug construction, nature communications, 2024, 15(1): 2831.
  5. Sun H., et al., Derivatization based on tetrazine scaffolds: synthesis of tetrazine derivatives and their biomedical applications. Organic Chemistry Frontiers. 2022, 9(2): 481-498.
  6. Carlson J C T., et al., Unraveling tetrazine-triggered bioorthogonal elimination enables chemical tools for ultrafast release and universal cleavage. Journal of the American Chemical Society. 2018, 140(10): 3603-3612.

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