[18F]insulin for PET imaging applications: simple and chemoselective two step reaction based on combined microfluidic-vessel chemistry

Aim: In vivo study of molecular events involved in deregulation of insulin signaling might be of high potential for early diagnosis and treatment of different metabolic and neurodegenerative disorders. PET imaging plays a pivotal role in this field due to its high sensitivity, noninvasiveness and quantification capability. Many approaches have been used for insulin labeling with positron emitting radionuclides. However, they have suffered from many disadvantages such as low yields and complex synthetic procedures. Click chemistry is a novel approach for radiolabeling of protein with PET radionuclides. The aim of our study is to describe a simple click chemistry procedure to synthesize a new [18F]insulin derivative for in vivo PET imaging applications. Methods: On the bases of the well documented insulin Structure-Relation-Activities (SAR) we functionalized the insulin PheB1 residue with a COOH (PEG)4-azido spacer. As model reaction the azido-protein was selectively reacted with a previously synthetized 19F-pentyne through Cu(I)-catalized cyclo addition (CuAAC). The 19F-insulin and reaction intermediates were characterized by HPLC and MALDI-TOF. The [18F]insulin was developed by a two step reaction based on the combination of microfluidic and vessel chemistry. 5-[18F]fluoro-1-pentyne was synthesized from the corresponding tosylate in a microfluidic system (ADVION) employing a not dried [18F]KF/Kryptofix complex in ACN at 130°C. The resulting intermediate was distilled and collected directly into a vial containing the azido-insulin derivative, Cu(I) and TBTA. The click reaction was performed at 45°C for 10 minutes and the final product was purified by RP-HPLC. Results: [19F]insulin was obtained with an overall reaction yield of 25% and in 95% purity. Product identity was confirmed by MALDI-TOF analysis. 5-[18F]fluoro-1-pentyne was obtained in 70% yield high yield and purity, as confirmed by radio HPLC and TLC. The [18F]insulin derivative was obtained with 65% yield and 95% purity as showed by analytical radio HPLC and confirmed by coinjection with the cold standard. In vivo imaging experiments for the assessment of the [18F]insulin tracer kinetics and biodistribution as well as the biological activity of the relative cold compound are currently in progress. Conclusion: A simple and efficient procedure for insulin radiolabeling was set up by combining vessel click chemistry and microfluidic 5-[18F]fluoro-1-pentyne radiosynthesis.