Synergistic Experimental and Computational Investigation of the Bioorthogonal Reactivity of Substituted Aryltetrazines.

Tetrazines (Tz) have been applied as bioorthogonal agents for various biomedical applications, including pretargeted imaging approaches. In radioimmunoimaging, pretargeting increases the target-to-background ratio while simultaneously reducing the radiation burden. We have recently reported a strategy to directly 18F-label highly reactive tetrazines based on a 3-(3-fluorophenyl)-Tz core structure. Herein, we report a kinetic study on this versatile scaffold. A library of 40 different tetrazines was prepared, fully characterized, and investigated with an emphasis on second-order rate constants for the reaction with trans-cyclooctene (TCO). Our results reveal the effects of various substitution patterns and moreover demonstrate the importance of measuring reactivities in the solvent of interest, as click rates in different solvents do not necessarily correlate well. In particular, we report that tetrazines modified in the 2-position of the phenyl substituent show high intrinsic reactivity toward TCO, which is diminished in aqueous systems by unfavorable solvent effects. The obtained results enable the prediction of the bioorthogonal reactivity and thereby facilitate the development of the next generation of substituted aryltetrazines for in vivo applications.

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

Pretargeted imaging of nanomedicines have attracted considerable interest because it has the potential to increase imaging contrast while reducing radiation burden to healthy tissue. Currently, the tetrazine ligation is the fastest bioorthogonal reaction for this strategy and, consequently, the state-of-art choice for in vivo chemistry. We have recently identified key properties for tetrazines in pretargeting. We have also developed a method to 18F-label reactive tetrazines using an aliphatic nucleophilic substitution strategy. Here, we combined this knowledge and developed an 18F-labeled tetrazine for pretargeted imaging. In order to develop this ligand, a small SAR study was performed. The most promising compound was selected for labeling and subsequent positron-emission-tomography in vivo imaging. Radiolabeling was achieved in satisfactory yields, molar activities, and high radiochemical purities. [18F]15 displayed favorable pharmacokinetics and remarkable target-to-background ratios-as early as 1 h post injection. We believe that this agent could be a promising candidate for translation into clinical studies.

Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted In Vivo Chemistry.

The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted in vivo chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile in vivo chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the in vivo ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the in vivo fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted trans-cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their in vivo performance. In particular, high rate constants (>50 000 M-1 s-1) for the reaction with TCO and low calculated logD 7.4 values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected 18F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for in vivo application and will thereby assist the clinical translation of bioorthogonal pretargeting.

Synthesis, structural characterization and biological evaluation of 4'-C-methyl- and phenyl-dioxolane pyrimidine and purine nucleosides.

Nucleoside analogues play an important role in antiviral, antibacterial and antineoplastic chemotherapy. Herein we report the synthesis, structural characterization and biological activity of some 4'-C -methyl- and -phenyl dioxolane-based nucleosides. In particular, α and ß anomers of all natural nucleosides were obtained and characterized by NMR, HR-MS and X-ray crystallography. The compounds were tested for antimicrobial activity against some representative human pathogenic fungi, bacteria and viruses. Antitumor activity was evaluated in a large variety of human cancer cell-lines. Although most of the compounds showed non-significant activity, 23α weakly inhibited HIV-1 multiplication. Moreover, 22α and 32α demonstrated a residual antineoplastic activity, interestingly linked to the unnatural α configuration. These results may provide structural insights for the design of active antiviral and antitumor agents.