Labelling of Proteins with Fluorine-18 via Click Chemistry

Objectives: The radiolabeling of peptides and proteins with the short-lived positron emitter ¹⁸F requires rapid and mild reaction conditions compatible with the structural and functional integrity of these biomolecules. Over the last two years several approaches have been published focusing on the application of copper(I)-mediated 1,3-dipolar [3+2]cycloaddition of azides and alkynes for labeling peptides with ¹⁸F. However, to date no ¹⁸F labeling of proteins via click chemistry has been reported. In this work we describe for the first time the application of click chemistry for ¹⁸F labeling of proteins as exemplified with azide-functionalized human serum albumin (HSA). Click chemistry was accomplished through 4-[¹⁸F]fluoro-N-methyl-N-(prop-2-ynyl)benzenesulfonamide (p[¹⁸F]F-SA) as novel alkine-containing ¹⁸F-labeled click chemistry building block.
Methods: The novel click chemistry building block p[¹⁸F]F-SA was prepared in a single step reaction in a remotely controlled synthesis module starting from readily available labeling precursor (Fig. 1).
HSA was modified with azide residues through conjugation of the lysine residues in HSA with an azide-functionalized active ester. Azide-modified HSA was subjected to digest with three different endoproteinases and subsequent MALDI-TOF MS analysis to assess the number of introduced azide residues. Radiolabeling of modified HSA was accomplished with p[¹⁸F]F-SA in the presense of Cu(I)Br and the Cu(I) chelating ligand tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). Radiolabeled HSA was purified with size-exclusion chromatography and analyzed with SDS-PAGE.
Results: Radiolabeled sulfonamide p[¹⁸F]F-SA could be obtained in an automated synthesis unit in radiochemical yields of 21-35% (decay-corrected) within 75 min after HPLC purification. The radiochemical purity was >99%, and the specific activity was in the range of 71-128 GBq/μmol. Sulfonamide p[¹⁸F]F-SA showed favorable lipophilicity (logP = 1.6) allowing application in aqueous reaction media. Tryptic digest and subsequent MALDI-TOF MS analysis of modified HSA revealed the introduction of an average of 28 azide residues into HSA. Click chemistry of azide-functionalized HSA (0.5 mg) with CuBr (0.2 mg) and TBTA in phosphate buffer (pH 7.4) gave 31% of ¹⁸F-labeled HSA after size-exclusion chromatography.
Conclusions: The convenient radiosynthesis of p[¹⁸F]F-SA as a novel 18F-labeled sulfonamide-based click chemistry building block in an automated synthesis unit allows its wide application for a broad range of click chemistry reactions. For the first time, click chemistry could successfully be applied to the ¹⁸F labeling of proteins, which further expands the scope of click chemistry as versatile tool for radiolabeling reactions.