¹⁸F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor (SiFA) for Positron Emission Tomography

The overall goal of the following procedure is to demonstrate the labeling of a ligand with the radionuclide fluorine-18, or F-18, using silicon fluoride acceptor compounds, hereafter referred to as SiFAs. Labeling a ligand with F-18 and injecting it into the body enables the imaging of numerous diseases with positron emission tomography, or PET. Fluorine-18 is among the most important radionuclides for PET imaging.

It has a half-life of 109 minutes, and 97%of its decay is by positron emission, making it nearly perfect for PET imaging. Peptides and proteins are especially difficult to label with F-18 because they require building blocks formed by multi-step synthesis. Otherwise, macromolecules will constitute a large proportion of available PET tracers.

To reduce the complexity of F-18 radiolabeling, silicon fluoride acceptors were introduced as a reliable tool. The SiFA group consists of a central silicon atom connected to two tertiary butyl groups, a derivatized phenyl moiety, and a fluorine atom. The two tertiary butyl groups impart hydrolytic stability to the silicon fluoride bond, which is a critical feature for in-vivo applications of SiFA conjugates as imaging agents.

The phenyl moiety acts as a linker between the radionuclide and biologically active molecule, represented as R in this diagram. When attached to a biomolecule, the SiFA building blocks readily exchange fluorine-19 for radioactive fluorine-18 due to the low activation energy of the isotopic exchange reaction. The labeling precursor and F-18 labeled compound are chemically identical, which makes purification relatively simple.

The non-canonical isotopic exchange labeling technique used with SiFA is also on par for ease of labeling with other novel labeling techniques such as trifluoroborate isotopic exchange and aluminum fluoride chelation. One must keep in mind that F-18 is a radioactive isotope. Therefore, it is necessary to carry out all procedures behind adequate shielding.

Lead shielding is appropriate for this type of radiation. Be sure to wear radiation detection badges throughout the entirety of this procedure. Additionally, immediately dispose of gloves before touching anything after the synthesis as they may be contaminated with radioactivity.

Utilize hand/foot monitors as well as pancake Geiger counters to check for contamination of sleeves, hands, and feet. A significant number of small organic molecules have been labeled with fluorine-18. However, interventional organic chemistry typically requires harsh reaction conditions to incorporate F-18 into these molecules.

These conditions are usually incompatible with sensitive chemical functionalities and delicate macromolecules such as proteins and peptides. Therefore, novel methods of labeling these molecules are highly sought after. The main advantage of the SiFA technique is that it allows a complicated procedure to be completed within one or two steps with minimal purification so that F-18 radio-pharmaceuticals may be prepared with less training and experience.

The implications of this technique extend to research groups interested in the in-vivo applications of experimental biomolecules. The SiFA labeling methodology utilizes F-18 fluoride, which is routinely produced in cyclotron facilities via the proton bombardment of O-18 water. For most F-18 reactions, the F-18 fluoride must first be separated from the cyclotron water to prepare an anhydrous organic stock solution of highly nucleophilic F-18 anions for reaction.

This is commonly achieved through the trapping of F-18 fluoride on an anion exchange cartridge, eluting the fluoride with a base and cryptand dissolved in acetonitrile and azeotropically drying the resulting solution before re-suspension in the dry reaction solvent of choice. Start by preconditioning a QMA exchange cartridge with 5 molar potassium carbonate followed by deionized water. Next, pass an aqueous solution of F-18 fluoride through the preconditioned QMA cartridge in reverse using a male-to-male adapter as shown.

If large amounts of radioactivity are to be handled, these steps can be performed using an automated synthesis module or by using additional shielding on the syringe. Discard the oxygen-18 water. Elute the first four drops of the fixed F-18 anions from the QMA cartridge with a prepared solution of kryptofix, potassium carbonate, and acetonitrile into a thick-walled V-vial and seal the vial.

Place the vial in an oil bath heated to 90 degrees. Only the first four drops are used as the majority of radioactive fluoride is eluted off the QMA with these drops. This reduces the amount of base carried forward into our F-18 stock solution, which is necessary to avoid degradation of the SiFA moiety.

Insert a vent needle and a needle connected to a stream of argon gas, then wait five minutes to evaporate the solvents. To remove traces of water, add acetonitrile to facilitate azeotropic evaporation. Complete this step twice to ensure dryness.

After removing the argon and vent needles, re-suspend the dry fluoride residue in the solvent of choice, in this case acetonitrile, to create a stock solution of highly reactive F-18. This solution can now be used for labeling. Precondition a C18 light cartridge by rinsing it with ethanol and with distilled water.

Add the F-18 fluoride stock solution to a reaction vial containing a SiFA labeled precursor. The entire stock solution can be added or an aliquot, depending on how much activity is desired for reaction. Allow the reaction to proceed for five minutes at room temperature without stirring.

Drop the reaction mixture into a syringe containing 1 molar phosphate buffer. Pass the solution through the preconditioned C18 cartridge to trap the labeled tracer. Next, wash the cartridge with distilled water, then elute the tracer with ethanol.

Dilute with sterile phosphate buffer for injection. Pass the resulting purified F-18 labeled tracer through a sterile filter. To obtain a clear PET image for small animal imaging, the partitioned patient dose should be between five to eight megabecquerel.

For human use, the partitioned patient dose should be between 200 to 300 megabecquerel. Gather and inject a small aliquot of the F-18 labeled tracer into an HPLC system equipped with a reversed-phase C18 column to confirm that the radiochemical purity is greater than 95%The F-18 labeled SiFA tracer can be injected into an animal or human subject, and the dynamic data collected from a PET scanner can be reconstructed to create a time series of three-dimensional PET images. In one example, this SiFA-tagged small molecule tracer developed for prostate cancer imaging has been successfully utilized to visualize tumors implanted in mice for preclinical studies.

The SiFA tag has also been shown to be suitable for radiolabeling, cancer targeting peptides such as tyrosine-3-octreotate or TATE. The F-18 labeled SiFAlin-TATE tracer shown here has been used to visualize neuroendocrine tumors in cancer patients. SiFA labeling chemistry alongside trifluoroborates and aluminum fluoride chelates represents one of the first F-18 labeling methods employing extraordinarily efficient isotopic exchange reaction at room temperature or below.

The method can be completed in as little as 30 minutes, which, compared to conventional radiolabeling procedures, minimizes chemical consumption and radioactive exposure.