## 9 Protein Labeling
Three different proteins, varying in molecular weight (66-144 kDa), were labeled in radiochemical yields between 30-80%, depending on the SiFA/protein ratio. RSA was again chosen to serve as an _in vivo_ blood pool imaging agent for PET. One shortcoming of this SiFA synthon is its inherent tendency towards hydrolysis and therefore associated challenging storage. The compound has to be meticulously stored under protecting gas and any exposure to humidity has to be avoided. Nevertheless, the use of isothiyanates omits the necessity of modifying the protein but rather depends on lysine residues in the protein. Figure 14: Radioactive labeling of RSA and other proteins (not shown) using (**A**) [\({}^{18}\)F]SiFA-isothiocyanate ([\({}^{18}\)F]SiFA-ITC, **[\({}^{18}\)F]**7), (**B**) [\({}^{18}\)F]SiFB and (**C**) the non-silicon containing most frequently applied protein labeling synthon [\({}^{18}\)F]SFB. (**D**) The PET images show the biodistribution of [\({}^{18}\)F]SFB (a and a’) and [\({}^{18}\)F]SiFB (b and b’) labeled RSA in rats (coronal plane sum images t = 5–60 min). The biodistribution was identical, validating the usefulness of [\({}^{18}\)F]SiFB as an alternative labeling agent for proteins. As already mentioned, by far the most common \({}^{18}\)F-labeling agent for proteins is [\({}^{18}\)F]SFB. In contrast to \({}^{18}\)F-labeled SiFA active esters, the preparation of non-radioactive SiFA active esters has never posed a problem. The Si-F bond is stable under a great variety of reaction conditions and an active ester moiety, such as a succinimidyl ester, can be introduced without problems. The challenge, however, is to undertake the isotopic exchange reaction without disintegration of the highly reactive active ester moiety. The SiFA labeling so far described uses a highly basic labeling cocktail containing large amounts of carbonate ions in addition to \({}^{18}\)F\({}^{-}\). Although not being an exceptionally good nucleophile under aqueous conditions, \({}^{18}\)F\({}^{-}\), in the presence of carbonate dissolved with Kryptofix2.2.2 to complexate the counter ion in dipolar aprotic solvents, is remarkably reactive. Under common labeling conditions, the [\({}^{18}\)F]SiFA active esters could not be detected, either on radio-TLC, or on HPLC, but only the hydrolyzed products. Very recently, [PERSON] and co-workers devised a modified composition of the \({}^{18}\)F-labeling solution. Strictly speaking, the isotopic exchange reaction on SiFAs does not mechanistically require a basic labeling environment. Thus, their hypothesis was that the basic \({}^{18}\)F-containing reaction mixture in acetonitrile can be simply neutralized by addition of a suitable acid. Indeed, the addition of oxalic acid to the labeling cocktail proved to be highly efficient and the isotopic exchange using SiFB (Figure 14B) proceeded without hydrolysis of the active ester moiety. [\({}^{18}\)F]SiFB is very similar to the already mentioned SiFA active esters (_cf._ Figure 3). For stability reasons, the ester moiety was placed _meta_ to the silicon atom, instead of _para_. The amount of oxalic acid added proved to be crucial. In all experiments, the less basic potassium oxalate had been used instead of carbonate and it was found that a ratio of oxalate/oxalic acid of 2:1 yielded the highest isotopic exchange yields. The reduction in complexity of synthesis and purification of [\({}^{18}\)F]SiFB in comparison to [\({}^{18}\)F]SFB was enormous. Depending on the [\({}^{18}\)F]SiFB synthesis, if either a full batch of \({}^{18}\)F\({}^{-}\) or just an aliquot of \({}^{18}\)F\({}^{-}\) was used, 25-50 min of reaction time could be saved. No HPLC purification had to be performed. The synthesis merely requires a reaction vial and a solid phase extraction cartridge. The subsequent protein labeling was as efficient as that using [\({}^{18}\)F]SFB. [\({}^{18}\)F]SiFB, and [\({}^{18}\)F]SFB labeled RSA were injected into healthy rats.