Radiolabelling of peptides with tetrazine ligation based on the inverse electron-demand Diels-Alder reaction: rapid, catalyst-free and mild conversion of 1,4-dihydropyridazines to pyridazines.

Click chemistry reactions, such as the tetrazine ligation, based on the inverse-electron demand Diels-Alder (IEDDA), are chemoselective cycloaddition reactions widely used for chemical modifications and synthesis of biomolecule-based radiopharmaceuticals for positron emission tomography (PET). The reactions have potential also for pretargeted PET imaging. When used as a bioconjugation method in production of biomolecule-based radiopharmaceuticals, IEDDA-based tetrazine ligation has one significant drawback, namely the formation of a mixture comprising reduced metastable dihydropyridazines (DHPs) and oxidized cycloadducts. Conversion of the reduced DHPs to stable pyridazines requires oxidation, which is typically achieved by using oxidants or by photo-irradiated air-oxidation, both methods requiring added reagents or reaction times of several hours, not compatible with short-lived radionuclides. Here we report a mild, rapid, and catalyst-free conversion of the DHPs to pyridazines. In this study, a model peptide Tyr3-octreotide (TOC) was modified with polyethylene glycol (PEG) linkers and with trans-cyclooctenes (TCOs) for rapid IEDDA-mediated radiolabeling. Fluorine-18-labelled alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazines were conjugated to the TCO-TOC analogs at room temperature for rapid synthesis of PET imaging agent candidates. The formed DHPs were successfully converted to the oxidized form, after heating the radiolabelled bioconjugates in aqueous solution (≥95% water) at 60 °C for a minimum of 10 minutes in the presence of air, resulting in one-pot back-to-back IEDDA reaction and DHP conversion. The water content of the reaction mixture was to be found critical for the coversion. Our finding offers a straightforward method for conversion of the metastable DHPs from the IEDDA-based tetrazine ligation to stable, oxidized pyridazines. The method is especially suitable for applications requiring rapid conversion.

Development of [18F]AmBF3 Tetrazine for Radiolabeling of Peptides: Preclinical Evaluation and PET Imaging of [18F]AmBF3-PEG7-Tyr3-Octreotide in an AR42J Pancreatic Carcinoma Model.

Radiolabeled peptides have emerged as highly specific agents for targeting receptors expressed in tumors for therapeutic and diagnostic purposes. Peptides developed for positron emission tomography (PET) are typically radiolabeled using prosthetic groups or bifunctional chelators for fast "kit-like" incorporation of the radionuclide into the structure. A novel [18F]alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazine (Tz), [18F]AmBF3-Tz, was developed for the [18F]fluorination of trans-cyclooctene (TCO)-modified biomolecules using Tyr3-octreotides (TOCs) as model peptides. [18F]AmBF3-Tz (Am = 15.4 ± 9.2 GBq/µmol, n = 14) was evaluated in healthy mice by ex vivo biodistribution and PET/computed tomography (CT), where the radiolabel in the prosthetic group was found stable in vivo, indicated by the low bone uptake in tibia (0.4 ± 0.1% ID/g, t = 270 min). TCO-TOCs tailored with polyethylene glycol (PEG) linkers were radiolabeled with [18F]AmBF3-Tz, forming two new tracers, [18F]AmBF3-PEG4-TOC (Am = 2.8 ± 1.8 GBq/µmol, n = 3) and [18F]AmBF3-PEG7-TOC (Am of 6.0 ± 3.4 GBq/µmol, n = 13), which were evaluated by cell uptake studies and ex vivo biodistribution in subcutaneous AR42J rat pancreatic carcinoma tumor-bearing nude mice. The tracer demonstrating superior behavior ex vivo, the [18F]AmBF3-PEG7-TOC, was further evaluated with PET/CT, where the tracer provided clear tumor visualization (SUVbaseline = 1.01 ± 0.07, vs SUVblocked = 0.76 ± 0.04) at 25 min post injection. The novel AmBF3-Tz demonstrated that it offers potential as a prosthetic group for rapid radiolabeling of biomolecules in mild conditions using bioorthogonal chemistry.

Metabolism of a Bioorthogonal PET Tracer Candidate [19F/18F]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS.

Organofluorosilicon based 18F-radiolabeling is an efficient method for incorporating fluorine-18 into 18F-radiopharmaceuticals for positron emission tomography (PET) by 19F/18F isotopic exchange (IE). The first PET radiopharmaceutical, 18F-SiFAlin-TATE, radiolabeled with a silicon-based [18F]fluoride acceptor (SiFA), namely, a para-substituted di-tert-butyl[18F]fluorosilylbenzene, has entered clinical trials, and is paving the way for other potential [18F]SiFA-labeled radiopharmaceuticals for diagnostic use. In this study, we report the in vitro metabolism of an oxime-linked SiFA tetrazine (SiFA-Tz), a new PET-radiotracer candidate, recently evaluated for pretargeted PET imaging and macromolecule labeling. Metabolism of SiFA-Tz was studied in mouse liver microsomes (MLM) for elucidating its major biotransformation pathways. Nontargeted screening by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) was utilized for detection of unknown metabolites. The oxime bond between the SiFA and Tz groups forms two geometric (E/Z) isomers, which underwent the same biotransformations, but unexpectedly with different kinetics. In total, nine proposed metabolites of SiFA-Tz from phase I and II reactions were detected, five of which were defluorinated in MLMs, elucidating the metabolic pathway leading to previously reported defluorination of [18F]SiFA-Tz in vivo. Based on the HRMS studies a biotransformation pathway is proposed: hydroxylation (+O) to tert-butyl group adjacent to the silicon, followed by oxidative defluorination (+OH/-F) cleaving the fluorine off the silicon. Interestingly, eight proposed metabolites of a reduced dihydrotetrazine analogue, SiFA-H2Tz, from phase I and II reactions were additionally detected. To the best of our knowledge, this is the first reported comprehensive investigation of enzyme mediated metabolic pathway of tetrazines and para-substituted di-tert-butylfluorosilylbenzene fluoride acceptors, providing novel structural information on the biotransformation and fragmentation patterns of radiotracers bearing these structural motifs. By investigating the metabolism preceding defluorination, structurally optimized new SiFA compounds can be designed for expanding the portfolio of efficient 19F/18F isotopic exchange labeling probes for PET imaging.

Evaluation of Organo [18F]Fluorosilicon Tetrazine as a Prosthetic Group for the Synthesis of PET Radiotracers.

Fluorine-18 is the most widely used positron emission tomography (PET) radionuclide currently in clinical application, due to its optimal nuclear properties. The synthesis of 18F-labeled radiotracers often requires harsh reaction conditions, limiting the use of sensitive bio- and macromolecules as precursors for direct radiolabeling with fluorine-18. We aimed to develop a milder and efficient in vitro and in vivo labeling method for trans-cyclooctene (TCO) functionalized proteins, through the bioorthogonal inverse-electron demand Diels-Alder (IEDDA) reaction with fluorine-18 radiolabeled tetrazine ([18F]SiFA-Tz). Here, we used TCO-modified bovine serum albumin (BSA) as the model protein, and isotopic exchange (IE) (19F/18F) chemistry as the labeling strategy. The radiolabeling of albumin-TCO with [18F]SiFA-Tz ([18F]6), providing [18F]fluoroalbumin ([18F]10) in high radiochemical yield (99.1 ± 0.2%, n = 3) and a molar activity (MA) of 1.1 GBq/µmol, confirmed the applicability of [18F]6 as a quick in vitro fluorination reagent for the TCO functionalized proteins. While the biological evaluation of [18F]6 demonstrated defluorination in vivo, limiting the utility for pretargeted applications, the in vivo stability of the radiotracer was dramatically improved when [18F]6 was used for the radiolabeling of albumin-TCO ([18F]10) in vitro, prior to administration. Due to the detected defluorination in vivo, structural optimization of the prosthetic group for improved stability is needed before further biological studies and application of pretargeted PET imaging.

Radiolabeled Molecular Imaging Probes for the In Vivo Evaluation of Cellulose Nanocrystals for Biomedical Applications.

Cellulose nanocrystals (CNCs) have remarkable potential to improve the delivery of diagnostic and therapeutic agents to tumors; however, the in vivo studies on CNC biodistribution are still limited. We developed CNC-based imaging probes for the in vitro and in vivo evaluation using two labeling strategies: site-specific hydrazone linkage to the terminal aldehyde of the CNC and nonsite-specific activation using 1,1'-carbonyldiimidazole (CDI). The in vivo behavior of unmodified CNC, DOTA-CNC (ald.), and DOTA-CNC (OH) was investigated in healthy and 4T1 breast cancer mouse models. They displayed good biocompatibility in cell models. Moreover, the biodistribution profile and SPECT/CT imaging confirmed that the accumulation of 111In-labeled DOTA-CNC (ald.) and 111In-DOTA-CNC (OH) was primarily in hepatic, splenic, and pulmonary ducts in accordance with the clearance of nontargeted nanoparticles. The developed CNC imaging probes can be used to obtain information with noninvasive imaging on the behavior in vivo to guide structural optimization for targeted delivery.