The authors have no acknowledgements.

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.
<ol>
	<li>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).</li>
	<li>Pass an aqueous solution of [18F]F-/[18O]H2O (100&#8722;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.</li>
	<li>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 &#956;L, 10 &#956;mol), 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.</li>
	<li>Seal the vial and place in a 90 &#176;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.</li>
	<li>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.</li>
	<li>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&#8722;500 MBq). This solution can now be used for labeling.</li>
</ol>
2. One-step SiFA-ligand labeling
NOTE: Figure 2B shows a workflow graph of this procedure, which takes ~15 min.
<ol>
	<li>Precondition a C-18 cartridge (Table of Materials) by rinsing it with ethanol (10 mL) and distilled water (10 mL).</li>
	<li>Add the [18F-]fluoride stock solution to a reaction vial containing a SiFA-labeled precursor (100 &#956;L, 20&#8722;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.</li>
	<li>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.</li>
	<li>Wash the cartridge with distilled water (5 mL), then elute trapped tracer from the C-18 cartridge with ethanol (300 &#956;L), and dilute with sterile phosphate buffer for injection (3 mL).</li>
	<li>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&#8722;8 MBq. For human use, the partitioned patient dose should be between 200&#8722;300 MBq.</li>
	<li>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%.</li>
</ol>

<ol>
	<li>Wahl, R. L., Buchanan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and practice of positron emission tomography. , (2002).</li><li>W&#228;ngler, C., Schirrmacher, R., Bartenstein, P., W&#228;ngler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-chemistry+reactions+in+radiopharmaceutical+chemistry:+Fast+&amp;+easy+introduction+of+radiolabels+into+biomolecules+for+in+vivo+imaging.">Click-chemistry reactions in radiopharmaceutical chemistry: Fast &amp; easy introduction of radiolabels into biomolecules for in vivo imaging.</a> Current Medical Chemistry. 17, 1092-1116 (2010).</li><li>Schirrmacher, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=18F-labeling+of+peptides+by+means+of+an+organosilicon-based+fluoride+acceptor.">18F-labeling of peptides by means of an organosilicon-based fluoride acceptor.</a> Angewandte Chemie International Edition. 45, 6047-6050 (2006).</li><li>Kostikov, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxalic+acid+supported+Si-18F-radiofluorination:+One-step+radiosynthesis+of+N-succinimidyl+3-(di-tert-butyl[18F]fluorosilyl)benzoate+([18F]SiFB)+for+protein+labeling.">Oxalic acid supported Si-18F-radiofluorination: One-step radiosynthesis of N-succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for protein labeling.</a> Bioconjugate Chemistry. 23 (1), 106-114 (2012).</li><li>Cacace, F., Speranza, M., Wolf, A. P., Macgregor, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleophilic+aromatic+substitution;+kinetics+of+fluorine-18+substitution+reactions+in+polyfluorobenzenes.+Isotopic+exchange+between+18F-+and+polyfluorobenzenes+in+dimethylsulfoxide.+A+kinetic+study.">Nucleophilic aromatic substitution; kinetics of fluorine-18 substitution reactions in polyfluorobenzenes. Isotopic exchange between 18F- and polyfluorobenzenes in dimethylsulfoxide. A kinetic study.</a> Journal of Fluorine Chemistry. 21, 145-158 (1982).</li><li>Schirrmacher, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+p-(di-tert-butyl[18F]fluorosilyl)benzaldehyde+([18F]SiFA-A)+with+high+specific+activity+by+isotopic+exchange:+A+convenient+labeling+synthon+for+the+18F-labeling+of+N-amino-oxy+derivatized+peptides.">Synthesis of p-(di-tert-butyl[18F]fluorosilyl)benzaldehyde ([18F]SiFA-A) with high specific activity by isotopic exchange: A convenient labeling synthon for the 18F-labeling of N-amino-oxy derivatized peptides.</a> Bioconjugate Chemistry. 18, 2085-2089 (2007).</li><li>Kostikov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-(4-(di-tert-butyl[18F]fluorosilyl)benzyl)-2-hydroxy-N,N-dimethylethylammonium+bromide+([18F]SiFAN+Br-):+A+novel+lead+compound+for+the+development+of+hydrophilic+SiFA-based+prosthetic+groups+for+18F-labeling.">N-(4-(di-tert-butyl[18F]fluorosilyl)benzyl)-2-hydroxy-N,N-dimethylethylammonium bromide ([18F]SiFAN+Br-): A novel lead compound for the development of hydrophilic SiFA-based prosthetic groups for 18F-labeling.</a> Journal of Fluorine Chemistry. 132, 27-34 (2011).</li><li>W&#228;ngler, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kit-like+18F-labeling+of+proteins:+Synthesis+of+4-(di-tert-butyl[18F]fluorosilyl)benzenethiol+(Si[18F]FA-SH)+labeled+rat+serum+albumin+for+blood+pool+imaging+with+PET.">Kit-like 18F-labeling of proteins: Synthesis of 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FA-SH) labeled rat serum albumin for blood pool imaging with PET.</a> Bioconjugate Chemistry. 20, 317-321 (2009).</li><li>Iovkova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=para-Functionalized+aryl-di-tert-butylfluorosilanes+as+potential+labeling+synthons+for+18F+radiopharmaceuticals.">para-Functionalized aryl-di-tert-butylfluorosilanes as potential labeling synthons for 18F radiopharmaceuticals.</a> Chemistry. 15, 2140-2147 (2009).</li><li>W&#228;ngler, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+18F-labeling+of+carbohydrate-conjugated+octreotate-derivatives+containing+a+silicon-fluoride-acceptor+(SiFA):+In+vitro+and+in+vivo+evaluation+as+tumor+imaging+agents+for+positron+emission+tomography+(PET).">One-step 18F-labeling of carbohydrate-conjugated octreotate-derivatives containing a silicon-fluoride-acceptor (SiFA): In vitro and in vivo evaluation as tumor imaging agents for positron emission tomography (PET).</a> Bioconjugate Chemistry. 21, 2289-2296 (2010).</li><li>Ilhan, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-in-human+18F-SiFAlin-TATE+PET/CT+for+NET+imaging+and+theranostics.">First-in-human 18F-SiFAlin-TATE PET/CT for NET imaging and theranostics.</a> European Journal of Nuclear Medicine and Molecular Imaging. 46, 2400-2401 (2019).</li></ol>

The authors have nothing to disclose.

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 reaction participants that must be removed using laborious and time-consuming techniques such as HPLC. With the SiFA-labeling technique, the labeling precursor and 18F-labeled compound are chemically identical. Moreover, no side products are usually observed since the reaction proceeds under very mild conditions. These features make it possible to label more complicated molecules (i.e., proteins, free radical generators, metal-chelates, fluorophores, bioluminescent precursors) that may normally decompose or epimerize under more reactive conditions or elevated temperatures. Additionally, 18F-labeled SiFA-containing compounds can be purified quickly using simple solid-phase extraction techniques.
This labeling methodology utilizes the &#39;4 drop method&#39;, wherein only the first 4 drops of the basic elution solution are used when eluting trapped [18F]fluoride off of a QMA cartridge. This modification was made to reduce the amount of base in the [18F]fluoride stock solution as it would degrade the SiFA moiety if the [18F]fluoride stock contained all of the base from the elution solution. Previously, oxalic acid was added to the [18F]fluoride stock solution to reduce basicity, or a small aliquot of the stock was used instead of the entire solution, which was wasteful. The &#39;4 drop method&#39; represents the latest iteration of the SiFA-labeling protocol.
As the precursor molecule and the labeled final product are chemically identical, they cannot be separated from each other during purification and the molar activity of the final injectable is thus entirely dependent on the quantity of precursor used for the isotopic exchange. Having too high of a fraction of unlabeled precursor in the final solution will decrease the opportunity of the labeled SiFA-ligand to bind its intended molecular target due to competition with the unlabeled precursor. Thus, the molar activity is entirely dependent on the amount of precursor that is used for labeling. Typically, 20&#8722;100 nmol of precursor is required for reproducible labeling reactions, and as little as 5 nmol of precursor has been labeled successfully to achieve molar activities of 80 GBq/&#181;mol and higher.
Small molecules and peptides derivatized with the SiFA building block (e.g., SiFA-octreotate) can be labeled with [18F]fluoride in one step; however, SiFA labeling of proteins requires a two-step protocol. A small, highly reactive SiFA-prosthetic group (e.g., [18F]SiFB) has to be prepared and reacted with the given protein, and the labeled protein must then be purified by HPLC.
The SiFA-labeling methodology lends itself well to radiopharmaceutical kit syntheses as HPLC purification and extensive reaction manipulation are not typically needed. Simple &#39;shake and bake&#39; style kits with single patient dose quantities of a SiFA-ligand could be easily handled by radiopharmacy technicians&#8211;requiring a much smaller learning curve and time/labor cost than with an automated synthesis unit.

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 calculations6,7. 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.

<table><tbody><tr><td>[&lt;sup&gt;18&lt;/sup&gt;F]F&lt;sup&gt;-&lt;/sup&gt;/H&lt;sub&gt;2&lt;/sub&gt;[&lt;sup&gt;18&lt;/sup&gt;O]O</td><td>(Cyclotron produced)</td><td>-</td><td>-</td></tr><tr><td>[2.2.2]Cryptand</td><td>Aldrich</td><td>291110</td><td>Kryptofix 2.2.2</td></tr><tr><td>Acetonitrile anhydrous</td><td>Aldrich</td><td>271004</td><td>-</td></tr><tr><td>Deionized water</td><td>Baxter</td><td>JF7623</td><td>-</td></tr><tr><td>Ethanol, anhydrous</td><td>Commercial Alcohols</td><td></td><td>-</td></tr><tr><td>Potassium carbonate</td><td>Aldrich</td><td>209619</td><td>-</td></tr><tr><td>QMA cartridge</td><td>Waters</td><td>186004540</td><td>QMA SepPak Light (46 mg) cartridge</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C-18 cartridge</td><td>Waters</td><td>WAT023501</td><td>C-18 SepPak Light cartridge</td></tr><tr><td>C18 column</td><td>Phenomenex</td><td>00G-4041-N0</td><td>HPLC Luna C18 250 x 10 mm, 5 &amp;micro;m</td></tr><tr><td>HPLC</td><td>Agilent Technologies</td><td>-</td><td>HPLC 1200 series</td></tr><tr><td>micro-PET Scanner</td><td>Siemens</td><td>-</td><td>micro-PET R4 Scanner</td></tr><tr><td>Radio-TLC plate reader</td><td>Raytest</td><td>-</td><td>Radio-TLC Mini Gita</td></tr><tr><td>Sterile filter 0.22&amp;micro;m</td><td>Millipore</td><td>SLGP033RS</td><td>-</td></tr></tbody></table>

18f-labeling of radiotracers functionalized with a silicon fluoride acceptor (sifa) for positron emission tomography

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the radiochemist&#39;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 &#39;4 drop method&#39;, 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.

The simplistic SiFA isotopic exchange can achieve high a degree of radiochemical incorporation of [18F]fluoride (60&#8722;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 its radio peak greater than 95% total peak area (Figure 3). If radio-HPLC chromatogram reveals a significant formation of UV active or radioactive impurities, the precursor may not be stable under the mildly basic radiolabeling conditions. A dilute solution of oxalic acid dissolved in an organic solvent may be added to the [18F]fluoride stock solution prior to the addition to SiFA-precursor in an effort to lower the basicity; however, lowering the basicity too much will lessen the nucleophilicity of the [18F]fluoride anion. Thus, the molar amount of oxalic acid needed will have to be experimentally determined beforehand. Alternatively, the labeled SiFA-ligand can be purified by HPLC after step 2.3 if the formation of impurities is significant but manageable. Steps 2.4 and beyond will still be needed after HPLC purification in order to remove HPLC solvent from the purified labeled SiFA-ligand.
If the [18F]fluoride is not readily incorporated into SiFA-ligand, there may be issues with solubility and another aprotic solvent of choice may be used in place of acetonitrile for the reaction. Protic solvents, such as ethanol, have been used successfully but may require heating. Monitoring the reaction by radio-thin-layer chromatography (radio-TLC) can quickly aide in identifying the outcome of any changes made to the labeling protocol as unincorporated [18F]fluoride will adhere to the baseline on a standard silica TLC plate.
If the labeled SiFA-ligand passes through the C18 cartridge in step 2.3, as indicated by the bulk of the activity appearing in the eluted solution and not in the cartridge, then the phase of the cartridge used may have to be changed. Polar SiFA-ligands may need a larger C18 cartridge or a dual-phase cartridge containing a C18 resin with some hydrophilic characteristics.
Labeled SiFA-ligands can also be used for in vivo applications such as PET. For instance, the labeled small molecule [18F]SiFA-PSMA (Figure 4A) was used to image an implanted tumor xenograft in a mouse model (Figure 4B). The SiFA-tracer displayed favorable tumor uptake over 60 min, which could be blocked by a competitive inhibitor (Figure 4C). More impressively, the 18F-labeled peptide [18F]SiFAlin-TATE (Figure 5A) was used to image metastatic neuroendocrine tumors in a cancer patient via PET (Figure 5B) and PET/CT (Figure 5C)11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60623/60623fig1.jpg" /> 
Figure 1: Overview of the SiFA 18F-radiolabeling workflow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60623/60623fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60623/60623fig2.jpg" /> 
Figure 2: Typical SiFA 18F-radiolabeling protocol. (A) Azeotropic drying of [18F]fluoride. (B) SiFA labeling reaction and purification via solid-phase extraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60623/60623fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60623/60623fig3.jpg" /> 
Figure 3: Final radio-HPLC chromatogram after solid-phase extraction purification of [18F]SiFA-PSMA, taken for quality control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60623/60623fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60623/60623fig4.jpg" /> 
Figure 4: Application of labeled SiFA-ligands. (A) Structure of the [18F]SiFA-PSMA radiotracer. (B) Reconstructed image of a mouse carrying an LNCaP xenograft tumor over left shoulder, 60 min post injection (p.i.) with [18F]SiFA-PSMA. (C) Time-activity curves for [18F]SiFA-PSMA uptake in tumor and muscle tissues over 60 min, with or without prior administration of 300 &#181;g DCFPyL as a blocking agent. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60623/60623fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60623/60623fig5.jpg" /> 
Figure 5: Application of labeled SiFA-ligands. (A) Structure of the [18F]SiFA-TATE radiotracer. (B) Reconstructed PET image of a human cancer patient with metastatic endocrine tumors with [18F]SiFA-TATE. (C) PET/CT images of the same patient in the transverse and sagittal planes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60623/60623fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about 18F-Labeling of Radiotracers Functionalized with a Silicon Fluoride Acceptor SiFA for Positron Emission Tomography at JoVE.com

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

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.

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

The para-substituted di-tert-butylfluorosilylbenzene structural motif known as the silicon-fluoride acceptor (SiFA) is a useful tag in the ...

18f-labeling-radiotracers-functionalized-with-silicon-fluoride

Nanyang Technological University

Research

JoVE Journal

Chemistry

7.3K Views.  University of Alberta. Het algemene doel van de volgende procedure is het aantonen van de etikettering van een ligand met de radionuclide fluor-18, of F-18, met behulp van siliciumfluoride acceptor verbindingen, hierna sifa's genoemd. Het labelen van een ligand met F-18 en het injecteren van het in het lichaam maakt de beeldvorming van tal van ziekten met positron emissie tomografie, of PET. Fluor-18 is een van de belangrijkste radionucliden voor PET-beeldvorming.Het heeft een halvetijd van 109 minuten, en 9...

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Cancer Research

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

Positron Emission Tomography Imaging of Cell Trafficking: A Method of Cell Radiolabeling

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for various cancers. Despite significant progress having been made in these areas, there is still more to be learned to better understand the pharmacokinetics and pharmacodynamics of the administered therapeutic cells in the living system. For noninvasive, in vivo tracking of cells with positron emission tomography (PET), a novel [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN)-mediated cell radiolabeling method has been developed utilizing 89Zr (t1/2 78.4 h). The present protocol describes a [89Zr]Zr-DBN-mediated, ready-to-use, radiolabeling synthon for direct radiolabeling of variety of cells, including mesenchymal stem cells, lineage-guided cardiopoietic stem cells, liver regenerating hepatocytes, white blood cells, melanoma cells, and dendritic cells. The developed methodology enables noninvasive PET imaging of cell trafficking for up to 7 days post-administration without affecting the nature or the function of the radiolabeled cells. Additionally, this protocol describes a stepwise method for the radiosynthesis of [89Zr]Zr-DBN, biocompatible formulation of [89Zr]Zr-DBN, preparation of cells for radiolabeling, and finally the radiolabeling of cells with [89Zr]Zr-DBN, including all the intricate details needed for the successful radiolabeling of cells.

Presented here is a protocol to radiolabel cells with a positron emission tomography (PET) radioisotope, 89Zr (t1/2 78.4 h), using a ready-to-use radiolabeling synthon, [89Zr]Zr-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN). Radiolabeling cells with [89Zr]Zr-DBN allows noninvasive tracking and imaging of administered radiolabeled cells in the body with PET for up to 7 days post-administration.

Stem cell and chimeric antigen receptor (CAR) T-cell therapies are emerging as promising therapeutics for organ regeneration and as immunotherapy for ...

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, &#945;-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models.
This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 &#177; 5% (decay corrected at the end of synthesis, n &#62; 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol

Here, we describe the radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-tryptophan, a positron emission tomography imaging agent for studying tryptophan metabolism, using a one-pot, two-step strategy in a radiochemistry synthesis system with good radiochemical yields, high enantiomeric excess, and high reliability.

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor ...

Radiosynthesis and PET/CT Imaging of 68Ga-Labelled Nanobody in Xenograft Models

Radiosynthesis, Quality Control, and Small Animal Positron Emission Tomography Imaging of 68Ga-Labelled Nano Molecules

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical cancer research, necessitating meticulous attention to radiotracer synthesis, quality assurance, and in vivo injection protocols. This study presents a comprehensive workflow tailored to enhance the robustness and reproducibility of small animal PET experiments. The synthesis process in the radiochemistry laboratory using 68Ga is detailed, highlighting stringent quality control and assurance protocols for each radiotracer production. Parameters such as concentration, molar activity, pH, and purity are rigorously monitored, aligning with standards applicable to human studies. This methodology introduces streamlined syringe preparation and a custom-designed 30G cannula for precise intravenous injections into mice. Monitoring of animal health during scanning, including temperature and heart rate, ensures their well-being throughout the procedure. Dosages for PET and SPECT scans are predetermined to balance data acquisition with minimizing radiation exposure to animals and researchers. Similarly, CT scans employ pre-programmed settings to limit radiation exposure, especially pertinent in long-term studies assessing treatment effects. By optimizing these steps, the workflow aims to standardize procedures, reduce variability, and enhance the quality of small animal PET/SPECT/CT imaging. This resource provides valuable insights for researchers seeking to improve the accuracy and reliability of preclinical investigations in molecular imaging, ultimately advancing the field.

This paper describes the radiosynthesis, formulation, quality control of a new radiolabeled probe (i.e., 68Ga-labeled nanobody NM-02), and its use for small animal PET/CT imaging in a xenograft model.

Small animal Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) imaging techniques are crucial in preclinical ...

Efficient Synthesis of Carbon-11 Radiotracers Using the Loop Method Methylation

Enhancing Efficiency and Radiolabeling Yields of Carbon-11 Radioligands for Clinical Research Using the Loop Method

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, requiring an on-site cyclotron and radiochemistry group, as well as clinical staff trained to operate under the unique constraints of the carbon-11 radionuclide. This study examines the merits and advantages of a captive solvent &#39;loop method&#39; of radiolabeling four tracers with the carbon-11 radionuclide, producing the radioligands [11C]ER-176, [11C]MRB, [11C]mHED, and [11C]PiB.
The &#39;loop method&#39; is compared against the traditional reactor-based method of carbon-11 methylation in the course of synthesizing the same radiotracers on the identical automated platform. Further, a complete overview of the clinical research preparation of the [11C]ER-176 radiotracer is presented. As demonstrated by the production of [11C]ER-176, the captive solvent &#39;loop method&#39; of heterogeneous alkylation proved to be more efficient, with excellent radiochemical purity (99.6 &#177; 0.6%, n = 25), higher and more consistent radiochemical yield (end of synthesis (EOS) = 5.4 &#177; 2.2 GBq, n = 25) compared to the reactor method (EOS = 1.6 &#177; 0.5 GBq, n = 6), increased molar activity (loop method = 194 &#177; 66 GBq/&#181;mol, n = 25; reactor method = 132 &#177; 78 GBq/&#181;mol, n = 6), along with an average 5 min shorter reaction sequence.

This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the &#8220;loop method&#8221; [11C]methylation. We describe the procedure to synthesize [11C]ER-176, &#160;with data for three additional radiotracers: [11C]MRB, [11C]mHED, and [11C]PiB. The loop method provides an efficient synthesis with increased radiochemical yields against traditional reaction vessel [11C]methylation.

A successful positron emission tomography imaging program involving carbon-11 radiotracers demands fast, efficient, and reliable synthesis methods, ...

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential therapeutics and molecular imaging agents. Notably, when labeled with positron-emitting radioisotopes (e.g., Cu-64, Ga-68, or F-18), they can be used as probes for targeted imaging of many physiological and pathological processes.15-18 Therefore, significant effort has devoted to the synthesis and exploration of 18F-labeled biomolecules. Although there are elegant examples of the direct 18F-labeling of peptides,19-22 the harsh reaction conditions (i.e., organic solvent, extreme pH, high temperature) associated with direct radiofluorination are usually incompatible with fragile protein samples. To date, therefore, the incorporation of radiolabeled prosthetic groups into biomolecules remains the method of choice.23,24
N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB),25-37 a Bolton-Hunter type reagent that reacts with the primary amino groups of biomolecules, is a very versatile prosthetic group for the 18F-labeling of a wide spectrum of biological entities, in terms of its evident in vivo stability and high radiolabeling yield. After labeling with [18F]SFB, the resulting [18F]fluorobenzoylated biomolecules could be explored as potential PET tracers for in vivo imaging studies.1 Most [18F]SFB radiosyntheses described in the current literatures require two or even three reactors and multiple purifications by using either solid phase extraction (SPE) or high-performance liquid chromatography (HPLC). Such lengthy processes hamper its routine production and widespread applications in the radiolabeling of biomolecules. Although several module-assisted [18F]SFB syntheses have been reported,29-32, 41-42 they are mainly based on complicated and lengthy procedures using costly commercially-available radiochemistry boxes (Table 1). Therefore, further simplification of the radiosynthesis of [18F]SFB using a low-cost setup would be very beneficial for its adaption to an automated process.
Herein, we report a concise preparation of [18F]SFB, based on a simplified one-pot microwave-assisted synthesis (Figure 1). Our approach does not require purification between steps or any aqueous reagents. In addition, microwave irradiation, which has been used in the syntheses of several PET tracers,38-41 can gives higher RCYs and better selectivity than the corresponding thermal reactions or they provide similar yields in shorter reaction times.38 Most importantly, when labeling biomolecules, the time saved could be diverted to subsequent bioconjugation or PET imaging step.28,43 The novelty of our improved [18F]SFB synthesis is two-fold: (1) the anhydrous deprotection strategy requires no purification of intermediate(s) between each step and (2) the microwave-assisted radiochemical transformations enable the rapid, reliable production of [18F]SFB.

Microwave-assisted One-pot Synthesis of N-succinimidyl-4-[18F]fluorobenzoate [18F]SFB

A facile, one-pot synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) was developed based on a non-aqueous, three-step radiochemical process. Using microwave heating, the entire procedure can be completed in less than 30 min, or 60 min with further purification by preparative HPLC. The decay-corrected radiochemical yields (RCYs) were 35-5% (n > 30).

Biomolecules, including peptides,1-9 proteins,10,11 and antibodies and their engineered fragments,12-14 are gaining importance as both potential ...

Biology

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Medicine

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

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

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