Tetrazine-trans-cyclooctene ligation (TTCO ligation) for the rapid construction of 18F labeled probes

79 Objectives F-18 PET has a number of attributes that make it clinically attractive, including almost 100% positron efficiency, a very high specific radioactivity, and a short half-life of ~110 min. However, the short half-life of F-18 and the poor nucleophilicity of fluoride render it difficult to incorporate F-18 in complex molecules. Recently, the TTCO ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we reported the development of an extremely fast and reactive method for generating 18F labeled probes based on the TTCO-ligation. Methods The trans-cyclooctene nosylate was prepared using a flow reactor that continuously removes the trans-isomers through selective metal complexation. 18F-trans-cyclooctene was obtained through the reaction between the nosylate precursor and TBAF. The efficacy of TTCO ligation was then tested using 18F-labeled trans-cyclooctene and tetrazine/tetrazine-RGD compounds. Results 18F-trans-cyclooctene was obtained with 71% labeling yield and the identity was confirmed by HPLC co-injection. The conjugations between 18F-trans-cyclooctene and 3,6-di(2-pyridyl)-s-tetrazine was completed within 10 seconds of mixing at low concentrations (≥ 98%). 18F-trans-cyclooctene could also effectively label the tetrazine-RGD peptides for integrin αvβ3 imaging. The integrin αvβ3 receptor specificity was confirmed by blocking experiments. Conclusions A radiolabeling method for bioconjugation based on the TTCO-ligation is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations. We anticipate that the TTCO-ligation will provide the foundation for a new class of reliable methods for 18F-labeling of biomolecules in PET applications. Research Support This work was supported by NIH Grant Number P20 RR017716 from the COBRE Program of the NCRR, and by the USC Department of Radiology