Fig. 2. (A) HPLC-DAD (280 nm, for HPLC method, see ESI Table S2†) chromatogram of IEDDA conjugation reaction between compound 1 and peptide with increasing molar amounts of 4, yielding peptide conjugate 6. The unreacted compound 1 used in excess is detected at tR = 10 min. The analogues a and b of the peptide 6 are detected at tR = 20.2–20.6 min (a, b) accompanied by an additional peak at tR = 17 min (c), hypothetically of 6 as well. (B) Radio-HPLC chromatogram of HPLC purified [18F]6 at tR = 20.2–20.6 min, together with the chromatograms of HPLC separated proposed isomers a (red trace) and b (blue trace) of the radiopeptide. (C) Radio-HPLC chromatogram of [18F]6 incubated in different reaction conditions revealing two analogues [18F]6a and b at tR = 20.2–20.6 min, and a third [18F]6c at tR = 17 min. The optimal reaction conditions resulting in ≥97% of [18F]6c after incubation at 60 °C for 10 min with minimum of 75% of water and maximum of 25% ACN (20–60 μL total volume and 2–7.5 nmol of TCO-PEG7-TOC).
