7 Tetrazine-Based Cycloadditions in Click Chemistry

The spontaneous cycloaddition of tetrazines with a number of different dienophiles has become a powerful tool in chemical biology, in particular for the biocompatible conjugation and modification of (bio)molecules. The exceptional reaction kinetics made these bioorthogonal ligations the methods of choice for time-critical processes at very low concentrations, facilitating controlled molecular transformations in complex environments and even in vivo. The emerging concept of bond-cleavage reactions triggered by tetrazine-based cycloadditions enabled the design of diagnostic and therapeutic strategies. The tetrazine-triggered activation of prodrugs represents the first bioorthogonal reaction performed in humans, marking the beginning of the era of clinical translation of bioorthogonal chemistry. This chapter provides an overview of the synthesis and reactivity of tetrazines, their cycloadditions with various dienophiles, and transformations triggered by these reactions, focusing on reaction mechanisms, kinetics and efficiency, and selected applications.

4.2 Applications of SPAAC and SPANC in Life Sciences

The bioorthogonal, strain-promoted azide–alkyne cycloaddition (SPAAC) and the strain-promoted alkyne–nitrone cycloaddition (SPANC) reactions have been used for conjugation with high affinity and specificity. In contrast to the cytotoxic copper-catalyzed cycloaddition, both SPAAC and SPANC are inert in biological environments. This chapter reviews the developments and applications of SPAAC and SPANC in life sciences reported since 2004, when Bertozzi et al. published the first bioorthogonal reaction.

A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

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.

Isocyanides: Promising Functionalities in Bioorthogonal Labeling of Biomolecules

Isocyanides have drawn increasing attention in biological applications due to their attractive properties and unique reactivities, which can undergo various reactions, such as multicomponent reactions, α-addition reactions, [4 + 1] cycloaddition reactions, and the reaction scope keeps expanding. In addition to acting as reactants for the preparation of structurally interesting and diverse N-heterocycles or peptidomimetics, this type of functionality may be a good choice in the labeling and modulation of biomolecules due to the high biocompatibility and small size to minimize modifications on the parent molecule. It has been demonstrated that isocyanides can participate in biomolecule labeling through three strategies, including the two-component bioorthogonal reaction, multicomponent reaction, and metal chelation. Among them, the isocyanide-tetrazine reaction has been better studied recently, augmenting the potency of isocyanide as a bioorthogonal handle. This review will focus on the recent progress in isocyanide chemistry for labeling of biomolecules. Meanwhile, methods to introduce isocyano groups into biomacromolecules are also described to facilitate wider applications of this unique functionality.

Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging – Expanding the Bioorthogonal Tool Box

<p>Pretargeting imaging of nanomedicines have attracted considerable interest in nuclear medicine since it has the potential to increase imaging contrast while simultaneously reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction available for this strategy and consequently, the state-of-art choice for <i>in vivo</i>chemistry. We have recently identified key properties for tetrazines to be applied in pretargeting. We have also developed a method to ¹⁸F-label highly reactive tetrazines using an aliphatic nucleophilic substitution strategy.<a> In this study, we combined this knowledge and developed an ¹⁸F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small structure-property study was carried out. The most promising compound - with respect to reactivity, hydrophilicity and <i>ex vivo</i> blocking effect - was selected for labeling and subsequent PET <i>in vivo</i> imaging. Radiolabeling was achieved in satisfying radiochemical yields, molar activities as well as in high radiochemical purities. The tracer </a><a>displayed favorable pharmacokinetics and remarkable target-to-background ratios in pretargeted experiments - already one hour post injection.</a> We believe that the developed pretargeting imaging agent is a promising candidate for translation into clinical studies.</p>

Development of the First Aliphatic 18F-Labeled Tetrazine Suitable for Pretargeted PET Imaging – Expanding the Bioorthogonal Tool Box

<p>Pretargeting imaging of nanomedicines have attracted considerable interest in nuclear medicine since it has the potential to increase imaging contrast while simultaneously reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction available for this strategy and consequently, the state-of-art choice for <i>in vivo</i>chemistry. We have recently identified key properties for tetrazines to be applied in pretargeting. We have also developed a method to ¹⁸F-label highly reactive tetrazines using an aliphatic nucleophilic substitution strategy.<a> In this study, we combined this knowledge and developed an ¹⁸F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small structure-property study was carried out. The most promising compound - with respect to reactivity, hydrophilicity and <i>ex vivo</i> blocking effect - was selected for labeling and subsequent PET <i>in vivo</i> imaging. Radiolabeling was achieved in satisfying radiochemical yields, molar activities as well as in high radiochemical purities. The tracer </a><a>displayed favorable pharmacokinetics and remarkable target-to-background ratios in pretargeted experiments - already one hour post injection.</a> We believe that the developed pretargeting imaging agent is a promising candidate for translation into clinical studies.</p>