¹²⁵I-Tetrazines and Inverse-Electron-Demand Diels–Alder Chemistry: A Convenient Radioiodination Strategy for Biomolecule Labeling, Screening, and Biodistribution Studies

Abstract

A convenient method to prepare radioiodinated tetrazines was developed, such that a bioorthogonal inverse electron demand Diels-Alder reaction can be used to label biomolecules with iodine-125 for in vitro screening and in vivo biodistribution studies. The tetrazine was prepared by employing a high-yielding oxidative halo destannylation reaction that concomitantly oxidized the dihydrotetrazine precursor. The product reacts quickly and efficiently with trans-cyclooctene derivatives. Utility was demonstrated through antibody and hormone labeling experiments and by evaluating products using standard analytical methods, in vitro assays, and quantitative biodistribution studies where the latter was performed in direct comparison to Bolton-Hunter and direct iodination methods. The approach described provides a convenient and advantageous alternative to conventional protein iodination methods that can expedite preclinical development and evaluation of biotherapeutics.

Prosthetic groups for radioiodination and astatination of peptides and proteins: A comparative study of five potential bioorthogonal labeling strategies

“…Indeed, while TCO/Tz-IEDDA provided quantitative cycloaddition yields within few seconds, it resulted in two dihydropyrazine tautomers that converted spontaneously within several hours to the corresponding aromatic pyrazine (see representative radiochromatograms in ESI), a phenomenon that has been reported previously with similar compounds. 16,23 This may raise concern regarding the lack of homogeneity of the resulting radiopharmaceutical and the consequence on pharmacokinetics, in vivo degradation of the conjugation bonds and on the carbon-radiohalogen bond stability. On the other hand, CuAAC, the second fastest system, provides the advantage of requiring fewer atoms than other systems (2 carbon and 3 nitrogen atoms), and resulting in a more hydrophilic product, a triazole core found in many naturally occurring biological compounds.…”

“…Click ligation of radioactive tags on various biomolecules are also the object of an important research effort to provide new strategies for labeling with radioisotopes that require fast, simple and efficient procedures that are difficult to reach by conventional chemistry. 12 Recent reports have proposed the use of bioorthogonal chemistry for radioiodination of peptides or proteins with clickable prosthetic groups including the strain-promoted alkyneazide cycloaddition (SPAAC) 13,14 and the inverse electron demand Diels Alder cycloaddition (IEDDA) reactions, 15,16 but to our knowledge, such strategy has not yet been reported for astatination. Yet, the necessity of fast, simple and quantitative yielding labeling procedure is even more essential for astatination with 211 At because of its availability in low amounts, its shorter physical half-life compared to relevant iodine radioisotopes, and the marked radiolysis issues due to the astatine decay limiting the starting activity that can be engaged in a radiolabeling procedure, 17 for the preparation of radiopharmaceuticals for clinical use, or even for animal studies.…”

Multifunctional Clickable Reagents for Rapid Bioorthogonal Astatination and Radio‐Crosslinking

“…In particular, tetrazine ligations – the fastest bioorthogonal reactions described so far – have had a significant impact by enabling strategies for (i) rapid radiolabeling using prosthetic groups, (ii) site‐specific radiolabeling, and (iii) pretargeting approaches based on in vivo chemistry . To this end, various radiolabeled 1,2,4,5‐tetrazines (Tz) have been developed as clickable tools for PET and SPECT imaging ( 18 F, 11 C, 111 In, 64 Cu, 123 I, 125 I), and targeted radiotherapy ( 177 Lu, 67 Cu) making use of the fast bioorthogonal ligation with trans ‐cyclooctenes (TCO).…”

Chemical synthesis of biomimetic hydrogels for tissue engineering

“…186–188 Among various dienenophiles reported so far, a conformationally strained trans -cyclooctene (sTCO) 189 is the fastest, with a rate up to 3.3 × 10 6 M 1 s −1 . With the unprecedented rate and tolerance to a broad range of biological functionalities, tetrazine ligation has become a broadly utilized bioorthogonal reaction, finding unique applications in cell labeling, 190–192 cancer imaging, 193, 194 and material science. 80, 195–197 …”