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

Summary

The synthesis of fluorine-18 (¹⁸F) labeled radiopharmaceuticals for positron emission tomography typically requires months of experience. When incorporated into a radiotracer, the silicon-fluoride acceptor (SiFA) motif enables a simple ¹⁸F-labeling protocol that is independent of costly equipment and preparatory training, while reducing precursor quantity needed and utilizing milder reaction conditions.

Abstract

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the radiochemist's toolkit for incorporating radioactive [¹⁸F]fluoride into tracers for use in positron emission tomography. In comparison to conventional radiolabeling strategies, isotopic exchange of fluorine-19 from SiFA with [¹⁸F]fluoride is carried out at room temperature and requires minimal reaction participants. The formation of by-products is thus negligible, and purification is greatly simplified. However, while the precursor molecule used for labeling and the final radiolabeled product are isotopically discrete, they are chemically identical and are thus inseparable during purification procedures. The SiFA tag is also susceptible to degradation under the basic conditions arising from the processing and drying of [¹⁸F]fluoride. The '4 drop method', wherein only the first 4 drops of eluted [¹⁸F]fluoride are used from the solid-phase extraction, reduces the amount of base in the reaction, facilitates lower molar amounts of precursor, and reduces degradation.

Introduction

Fluorine-18 (109-minute half-life, 97% positron emission) is among the most important radionuclides for positron emission tomography (PET), a noninvasive imaging method that visualizes and quantifies the bio-distribution of radiolabeled tracers for various diseases¹. Peptides and proteins are especially difficult to label with [¹⁸F]fluoride because they require building blocks formed by multi-step syntheses². To reduce the complexity of ¹⁸F-radiolabeling, silicon-fluoride acceptor (SiFA) was recently introduced as reliable tools³. The SiFA group consists of a central silicon atom connected to two tertiary butyl groups, a derivatized phenyl moiety, and a non-radioactive fluorine atom. The 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.

When attached to a small molecule or biomolecule, the SiFA building blocks bind radioactive [¹⁸F]fluoride anions by exchanging fluorine-19 for fluorine-18 at nanomolar concentrations without forming significant amounts of radioactive side products⁴. Moreover, a high radiochemical yield is quickly achieved by labeling the SiFA moiety in dipolar aprotic solvents at low temperatures. This is in stark contrast to classical isotopic exchange reactions, which produce radiotracers of low specific activity⁵. In these cases, large amounts of precursor (in the range of milligrams) must be used to obtain reasonable incorporation of [¹⁸F]fluoride. Isotopic exchange reactions using SiFAs are far more efficient, as confirmed by kinetic studies and density functional theory calculations^6,7. Labeled SiFAs are easily purified by solid-phase extraction since both the labeled and unlabeled SiFA compounds are chemically identical. This differs from traditional radiolabeled tracers, where the precursor molecule and the labeled product are two different chemical species and must be separated after radiolabeling by high-performance liquid chromatography (HPLC). Using SiFA building blocks, small-molecules, proteins, and peptides can be successfully labeled with [¹⁸F]fluoride by one- and two-step labeling protocols devoid of complicated purification procedures (Figure 1)^4,8,9. Moreover, some SiFA-labeled compounds are reliable in vivo imaging agents for blood flow and tumors¹⁰. The simplicity of SiFA chemistry enables even untrained investigators to use [¹⁸F]fluoride for radiotracer synthesis and development.

Protocol

CAUTION: One must keep in mind that ¹⁸F is a radioactive isotope, and 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 radioactive activity. Utilize hand-foot monitors as well as pancake Geiger counters to check for contamination of sleeves, hands, and feet.

1. Azeotropic drying of ¹⁸F-anion

NOTE: Figure 2A shows a workflow graph of this procedure, which takes ~10 min.

1. Precondition a quaternary methyl ammonium (QMA) anion exchange cartridge (Table of Materials) by passing 0.5 M K₂CO₃ (10 mL) through the cartridge, followed by deionized water (10 mL).
2. Pass an aqueous solution of [¹⁸F]F^-/[¹⁸O]H₂O (100−500 MBq) through the preconditioned QMA cartridge in reverse, using a male to male adapter. Discard the [¹⁸O]H₂O.
   NOTE: These steps can be performed using an automated synthesis module, or by using additional shielding on the syringe.
3. Elute the first four drops of the fixed [¹⁸F]fluoride anions from the QMA cartridge into a prepared solution of [2.2.2]cryptand (Table of Materials) (10 mg), 0.2 M K₂CO₃ (50 μL, 10 μmol), and acetonitrile (1 mL) in a thick-walled v-vial, and seal the vial.
   NOTE: Only the first four drops are used as the majority of the radioactive [¹⁸F]fluoride is eluted off the QMA in these drops. This reduces the amount of base carried forward in the [¹⁸F]fluoride stock solution, which is necessary to avoid degradation of the SiFA moiety.
4. Seal the vial and place in a 90 °C mineral oil bath positioned on a hot plate. Insert a vent needle and a needle connected to a stream of argon gas into the septum of the vial cap. Wait 5 min to evaporate the solvents under the gentle stream of argon. Remove any remaining traces of water by adding 1 mL of acetonitrile to facilitate azeotropic co-evaporation. Repeat this step 2x to ensure dryness.
5. Once the solvent is visibly removed, stop the argon flow, and remove the syringes from the vial cap, and remove the vial from the oil bath.
6. Resuspend the dried [¹⁸F]fluoride in the reaction solvent of choice.
   NOTE: In this case, acetonitrile (1 mL) is added to create a stock solution of highly reactive [¹⁸F^-]F^- (100−500 MBq). This solution can now be used for labeling.

2. One-step SiFA-ligand labeling

NOTE: Figure 2B shows a workflow graph of this procedure, which takes ~15 min.

1. Precondition a C-18 cartridge (Table of Materials) by rinsing it with ethanol (10 mL) and distilled water (10 mL).
2. Add the [¹⁸F^-]fluoride stock solution to a reaction vial containing a SiFA-labeled precursor (100 μL, 20−100 nmol). Allow the labeling reaction to proceed for 5 min at room temperature without stirring the solution.
   NOTE: The entire stock solution can be added or an aliquot, depending on how much activity is desired for the reaction.
3. Draw up the reaction mixture in a 20 mL syringe containing 0.1 M phosphate buffer (9 mL) and pass the solution through the preconditioned C-18 cartridge to trap the labeled tracer.
4. Wash the cartridge with distilled water (5 mL), then elute trapped tracer from the C-18 cartridge with ethanol (300 μL), and dilute with sterile phosphate buffer for injection (3 mL).
5. Pass the purified [¹⁸F]SiFA-tracer through a sterile filter.
   NOTE: To obtain a clear PET imagine for small animal imaging, the partitioned patient dose should be between 5−8 MBq. For human use, the partitioned patient dose should be between 200−300 MBq.
6. Inject a small aliquot (~4 MBq) of the purified [¹⁸F]SiFA-tracer onto an HPLC system equipped with a reversed-phase C-18 column to confirm that the radiochemical purity is greater than 95%.

Results

The simplistic SiFA isotopic exchange can achieve high a degree of radiochemical incorporation of [¹⁸F]fluoride (60−90%) with a minimum amount of synthetic complexity (Figure 1). Most molecules can be radiolabeled with [¹⁸F]fluoride in one step without involving HPLC for purification (Figure 2). Radio-HPLC can be used for quality control purposes, wherein the ultraviolet (UV) absorbance peak of the final product should coincide with i...

Discussion

SiFA labeling chemistry represents one of the first ¹⁸F-labeling methods employing an extraordinarily efficient isotopic exchange reaction that can be performed at room temperature. A typical radiochemical reaction relies on the formation of a carbon-fluorine bond via reaction of [¹⁸F]fluoride with a fluoride-reactive functionality through an elimination or substitution pathway. These reaction conditions are often harsh, performed at extreme pH or high temperature, and are laden with byproducts or r...

Materials

Name	Company	Catalog Number	Comments
[18F]F-/H2[18O]O	(Cyclotron produced)	-	-
[2.2.2]Cryptand	Aldrich	291110	Kryptofix 2.2.2
Acetonitrile anhydrous	Aldrich	271004	-
Deionized water	Baxter	JF7623	-
Ethanol, anhydrous	Commercial Alcohols		-
Potassium carbonate	Aldrich	209619	-
QMA cartridge	Waters	186004540	QMA SepPak Light (46 mg) cartridge
Equipment
C-18 cartridge	Waters	WAT023501	C-18 SepPak Light cartridge
C18 column	Phenomenex	00G-4041-N0	HPLC Luna C18 250 x 10 mm, 5 µm
HPLC	Agilent Technologies	-	HPLC 1200 series
micro-PET Scanner	Siemens	-	micro-PET R4 Scanner
Radio-TLC plate reader	Raytest	-	Radio-TLC Mini Gita
Sterile filter 0.22µm	Millipore	SLGP033RS	-