## 9 Protein Labeling  
The successful labeling of proteins with the radionuclide \({}^{18}\)F has always been challenging. Unlike peptides, which are structurally manageable, proteins contain a vast number of side chain functionalities which cannot be masked by protecting groups to enable a direct one-step \({}^{18}\)F labeling strategy. Hence, the labeling of proteins requires prosthetic groups, small molecules which are \({}^{18}\)F-labeled prior to the actual protein labeling. These molecules bear functional groups, such as active esters, which react readily with nucleophilic groups in the protein via acylation. [18]SFB is currently  
Figure 12: Structures of \({}^{18}\)F-SiFA-TATE **(29)**, \({}^{18}\)F-SiFA-Glc-TATE **(36)** and \({}^{18}\)F-SiFA-Glc-PEG-TATE **(37)**. the most common protein labeling agent. Besides active esters, Click Chemistry has also been employed, especially the maleimide-thiol click reaction. Of all Si-\({}^{18}\)F approaches, so far only the SiFA \({}^{19}\)F by \({}^{18}\)F-exchange chemistry has been used to develop small silicon-containing prosthetic groups for protein labeling. The first attempt to synthesize a SiFA-based active ester was made by [PERSON] and co-workers in 2009. The non-radioactive labeling precursors were obtained according to Figure 13 but could not be labeled with \({}^{18}\)F by isotopic exchange under basic labeling conditions. The active ester moiety was simply hydrolyzed during the isotopic exchange and no SiFA active ester could be isolated. Using a different strategy, rat serum albumin (RSA), a 64 kDa protein isolated from rat serum, was labeled with two different SiFA synthons, a maleimide- \({}^{18}\)F-SiFA ([\({}^{18}\)F]**11**) and a thiol- \({}^{18}\)F-SiFA ([\({}^{18}\)F]**4**) (Figure 13). In order to use the maleimide-SiFA, the protein was derivatized with 2-iminithiolane to provide additional thiol groups to react with the maleimide group of the SiFA synthon (Figure 13). In case of thiol-SiFA used for protein labeling, the protein was modified, complementary with sulfosuccinidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC). Both SiFA prosthetic groups [\({}^{18}\)F]**11** and [\({}^{18}\)F]**4** readily reacted with the modified RSA via Click Chemistry and provided the \({}^{18}\)F-labeled proteins in good yields. This method is very convenient, since the number of derivatization sites introduced at the protein modification step (maleimide or thiol introduction) can be easily determined by [PERSON]'s assay. The labeled RSA was used in small animal PET imaging, visualizing the blood pool. However, one adverse feature of this strategy, despite being one of the most convenient procedures described so far, is that the proteins have to be modified before the actual labeling occurs. This requires certain special knowledge from the user and could be deemed as being less comfortable. To resolve this inconvenience, [PERSON] and co-workers endeavored to mimic a well-known concept in bio-conjugation--the use of isothi cyanates for the modification and labeling of proteins. The goal of  
Figure 13: Kit-like labeling of RSA using two different SiFA sythons: A) sulfo-SMCC modified RSA reacts with thiol-SiFA ([\({}^{18}\)F]**4**); B) 2-iminothiolane modified RSA reacts with maleimide-SiFA ([\({}^{18}\)F]**11**). their study was to develop a radioactive counterpart to fluorescein isothiocyanate which is the most commonly applied fluorescent labeling agent for lysine containing proteins. Following that idea, SiFA isothiamate (SiFA-NCS or SiFA-ITC) was synthesized and \({}^{18}\)F-labeled at room temperature via isotopic exchange [45]. The amount of SiFA used was very low (10-12 nmol) and the radiochemical yields were surprisingly high, keeping in mind that the isothiamate moiety is very labile. After 10 min reaction time, 95% of the introduced \({}^{18}\)F\({}^{-}\) was incorporated into the SiFA-NCS. The \({}^{18}\)F-SiFA-NCS reacts with primary amino functions of lysine residues.