Peking University realizes supramolecular reversible regulation of tetrazine bioorthogonal reactions

On June 15, 2023, Professor Liu Tao of Peking University and Professor Jiang Wei’s research team from Southern University of Science and Technology published a new study entitled “Reversible control of tetrazine bioorthogonal reactivity by naphthotube-mediated host-guest recognition” in the journal Chem.

In this study, a bioorthogonal reactivity regulation strategy of tetrazine based on supramolecular host-guest recognition is reported, which regulates the bioorthogonal reactivity of tetrazine through molecular recognition of naphthalene tube subjects and tetrazine guests. Naphthalene tubes recognize phenyltetrazine on various biomolecules with high affinity, thereby effectively inhibiting their bioorthogonal reactivity in a reversible manner. This strategy is expected to expand the application of tetrazine chemistry.

The corresponding author of the paper is Professor Liu Tao; The first authors are Cao Wenbing, Wang Haoyu, Quan Mao.

Bioorthogonal chemical reactions refer to a class of chemical reactions that can be carried out in biological systems and do not interfere with natural biochemical processes. The emergence of such chemical reactions has brought revolutionary technologies to the in-situ study of life processes by scientists, and has become one of the core directions of the emerging interdisciplinary field of chemical biology. For this reason, the 2022 Nobel Prize in Chemistry was awarded to chemists Carolyn R. Bertozzi, Morten Meldal and K. Barry Sharpless in recognition of their contributions to the study of click chemistry and biological orthogonal chemistry. Among them, the Diels–Alder (IEDDA) reaction with the inverse electron demand involved in tetrazine has an ultra-fast reaction rate, mild reaction conditions and excellent biocompatibility, which is a very potential bioorthogonal reaction. The precise regulation of this reaction is also of great significance. However, at present, most of these regulatory means are limited to photocatalytic or electrochemical reduction of dihydrotetrazine, thereby releasing its reactivity, although effective regulation is achieved, it is toxic to organisms and inconvenient to operate.

In this work, Professor Liu Tao’s research group developed a non-covalent strategy for regulating the bioorthogonal activity of tetrazine, which is simple, efficient and biocompatible. The group previously found that supramolecular subjects can recognize guests in the form of amino acid side chains on the surface of proteins, thereby targeting proteins and affecting their functions (Angew. Chem. Int. Ed. 2021, 60, 11196), and Professor Wei Jiang’s group found that supramolecular naphthalene tubes can have high affinity with phenylpyrimidine (CCS Chem. 2020, 2, 1078–1092). Therefore, in this work, Professor Liu Tao’s group developed a strategy for supramolecular naphthalene tubes to recognize tetrazine and regulate its bioorthogonal reactivity (Figure 1). Theoretical calculations and experiments have verified that naphthalene tubes can have high affinity with phenyltetrazine, and the binding can effectively inhibit the reactivity of tetrazine. This molecular recognition is effective for different molecular modifications of tetrazine, including fluorescent molecules, sugars, amino acids, nucleic acids, and proteins. Moreover, the reactivity of caged tetrazine can be restored by adding competitive small molecules with high affinity with the naphthalene tubes. Therefore, this strategy can realize the reversible regulation of tetrazine bioorthogonal reactions on a variety of biological macromolecules.

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Figure 1: Schematic diagram of a supramolecular naphthalene tube regulating tetrazine bioorthogonal reaction.

By making tetrazine into the form of an amino acid side chain, tetrazine amino acids can be genetically encoded and participate in the translation of proteins, realizing site-specific tetrazine modification of proteins. Through theoretical calculations and experimental measurements, naphthalene tubes can only identify “standing” tetraazine amino acids that are fully exposed to solvents on the surface of the protein, but cannot identify “lying” tetraazine amino acids attached to the surface, and both of them have bioorthogonal reactivity. Therefore, the “standing” tetrazine amino acids can first undergo bioorthogonal reactions by caging the “standing” tetrazine amino acids, and then let the “standing” tetrazine amino acids participate in the new molecularly-mediated biological orthogonal reactions through the addition of competitive small molecules. This sequential IEDDA reaction enables fast-sited bioorthogonal reaction multi-modifications in proteins inserted with multiple tetrazine amino acids (Figure 2A).

In view of the widespread use of antibody-drug conjugates (ADCs) in clinical treatment and poor stability of antibody proteins, the mild and rapid IEDDA reaction is very suitable for the preparation of antibody small molecule conjugates. Therefore, such a sequential IEDDA reaction should first be applied to the gentle and rapid multilabeling of antibodies. By expressing antibodies containing multiple tetrazine amino acids and performing sequential IEDDA reactions, site-specific modified long-acting fluorescent antibodies and fluorescent antibody drug conjugates were prepared, and their biological activities were characterized. This suggests that the strategy of sequential IEDDA reactions can achieve precise multisite modification of antibodies and dual conjugation of antibody drug fluorescence (Figure 2B).

Due to its gentle and rapid characteristics, the tetrazine IEDDA reaction is widely used in the labeling of living cells. Supramolecular regulatory elements are also extremely biocompatible. By expressing a variety of tetrazine-containing amino acids in the cells themselves, sequential IEDDA reactions can enable site-directed labeling and efficient imaging of multiple targets within the same living cell (Figure 2C, D).

Tetrazine IEDDA has a high reaction rate and mild reaction conditions, which allows it to be used for reactions in vivo. Therefore, experiments on regulating tetrazine bioorthogonal reactivity in vivo in mice were also performed (Figure 2E, F).

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Figure 2: Application of supramolecular naphthalene tubes to regulate tetrazine bioorthogonal reactions.

In conclusion, this regulation strategy expands the application scenarios of tetrazine bioorthogonal chemistry and provides new inspiration for the development of tetrazine bioorthogonal chemistry. This work was supported by the National Natural Science Foundation of China (92156025, 92253301 and U22A20332), the National Key Research and Development Program of China (2022YFA0912400 and 2021YFA0909900), and the Beijing Natural Science Foundation (JQ20034). Team members Li Yuxuan, Su Yeyu, Li Yuhang, Zhang Xiaohui and Shi Xiaomeng, platform advisors of the State Key Laboratory of Natural Medicine and Biomimetic Drugs, and Zhao Yijie, instructor of the Department of Laboratory Animal Science, Peking University Health Science Center, also made important contributions to the work. (Source: Science Network)

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