Tetrazine Ligation Research Articles

Development and Evaluation of 18F-TTCO-Cys40-Exendin-4: A PET Probe for Imaging Transplanted Islets

Because islet transplantation has become a promising treatment option for patients with type 1 diabetes, a noninvasive imaging method is greatly needed to monitor these islets over time. Here, we developed an (18)F-labeled exendin-4 in high specific activity for islet imaging by targeting the glucagonlike peptide-1 receptor (GLP-1R). Tetrazine ligation was used to radiolabel exendin-4 with (18)F. The receptor binding of (19/18)F-tetrazine trans-cyclooctene (TTCO)-Cys(40)-exendin-4 was evaluated in vitro with INS-1 cell and in vivo on INS-1 tumor (GLP-1R positive) and islet transplantation models. (18)F-TTCO-Cys(40)-exendin-4 was obtained in high specific activity and could specifically bind to GLP-1R in vitro and in vivo. Unlike the radiometal-labeled exendin-4, (18)F-TTCO-Cys(40)-exendin-4 has much lower kidney uptake. (18)F-TTCO-Cys(40)-exendin-4 demonstrated its great potential for transplanted islet imaging: the liver uptake value derived from small-animal PET images correlated well with the transplanted β-cell mass determined by immunostaining. Autoradiography showed that the localizations of radioactive signal indeed corresponded to the distribution of islet grafts in the liver of islet-transplanted mice. (18)F-TTCO-Cys(40)-exendin-4 demonstrated specific binding to GLP-1R. This PET probe provides a method to noninvasively image intraportally transplanted islets.

Application of Strain-Promoted Azide–Alkyne Cycloaddition and Tetrazine Ligation to Targeted Fc-Drug Conjugates

We have previously described an approach whereby antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec) are directed against a variety of targets by changing the peptide or small molecule to which they are conjugated. In the present work, we describe methodology for improving the efficacy of these Fc-Sec conjugates by incorporating cytotoxic drugs. The Fc-Sec protein is first programmed to target specific tumor cell types by attachment of a bifunctional linker that contains a "clickable" handle (e.g., cyclobutane or cyclooctyne) in addition to a tumor cell-binding peptide or small molecule. Following Fc-Sec conjugation, a cytotoxic warhead is then attached by cycloaddition reactions of tetrazine or azide-containing linker. To validate this approach, we used a model system in which folic acid (FA) is the targeting moiety and a disulfide-linked biotin moiety serves as a cytotoxic drug surrogate. We demonstrated successful targeting of Fc-Sec proteins to folate-receptor expressing tumor cells. Tetrazine ligation was found to be an efficient method for biotin "arming" of the folate-targeted Fc-Sec proteins. We also report novel bioconjugation methodologies that use [4 + 2] cycloaddition reactions between tetrazines and cyclooctynes.

The Controlled Display of Biomolecules on Nanoparticles: A Challenge Suited to Bioorthogonal Chemistry

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.