We would like to acknowledge former NYULH Radiochemistry lab members, Raul Jackson and Grace Yoon, for their work on initial efforts for the carbon-11 methylation using a loop method.

NOTE: All equipment and syntheses are performed in either a lead-shielded hotcell or minicell. CAUTION: High-energy positron-emitting particles are generated and used from the cyclotron. The area is monitored with calibrated Geiger counters, and individuals wear radiation safety-issued dosimetry rings and badges. All personnel are trained to work with high-energy radioactive materials.
All syntheses are performed on a clean and sterilized Module according to our internal standard operating procedures (SOP). The cleaning process includes the use of 1 N HCl, water, acetone, and acetonitrile for the reaction portion of the Module. Meanwhile, the formulation portion of the Module is cleaned and sterilized with water and ethanol.
1. The radiolabeling of [&#160;11C]ER-176 (1) by way of loop method
NOTE: For a list of the materials used in preparing [11C]ER-176 (1), please see Table of Materials.
<ol>
	<li>Replumb the Module for the loop method production (see Figure 4A-D). Attach a V8 valve to a union that directly connects into the HPLC loop. This will bypass the reaction vessel.</li>
	<li>Condition the methane (CH4) furnace at 350 oC for 20 min with a hydrogen gas flow of 100 mL/min prior to all productions performed on the Module. Cool to 45 oC before proceeding.</li>
	<li>Condition the CH4 trap at 120 oC for 20 min with a helium gas flow of 50 mL/min prior to all productions performed on the Module. Cool to 70 oC before proceeding.</li>
	<li>Condition the methyl iodide trap at 190 oC for 20 min with a helium gas flow of 50 mL/min prior to all productions performed on the Module. Cool to 50 oC before proceeding.</li>
	<li>Reagent loading for the Module:
	<ol>
		<li>Using a 100 &#181;L syringe, inject the following mixture through the adapter at position #1 of the six-port valve&#8217;s internal HPLC loop: (R)-N-sec-butyl-4-(2-chlorophenyl)-quinazoline-2-carboxamide (1.0 &#177; 0.05 mg) in dimethyl sulfoxide-d6 (100 &#177; 10 &#956;L) and 6 N sodium hydroxide (4 &#956;L). 
		NOTE: Deuterated solvents were selected for their availability in ampule form and because they are typically distilled during manufacturing, ensuring they are free from water.</li>
		<li>Load the V4 reservoir with 3.0 mL of 0.9% sodium chloride for injection.</li>
		<li>Load the V5 reservoir with 1.0 mL of 200-proof ethanol.</li>
		<li>Load the V6 reservoir with 10 mL of sterile deionized water.</li>
		<li>Fill the large receiving flask with 25 mL of sterile deionized water.</li>
		<li>Fill the formulation flask with 6.0 mL of 0.9% sodium chloride for injection.</li>
		<li>Ensure the delivery line is attached to a sterile preassembled final product vial.</li>
		<li>Prepare the mobile phase with a 37:63 (v/v) mixture of acetonitrile and 20 mM ammonium hydroxide.</li>
		<li>Condition the semi-prep column with four column volumes of mobile phase. 
		NOTE: The column used is a C18 100 mm x 10 mm reverse-phase column.</li>
	</ol>
	</li>
	<li>During the preparation of the automated module, bombard carbon-11 cyclotron target (1% oxygen, 99% nitrogen) on an 11 MeV cyclotron at 55 &#956;A for 60&#8211;80 min on dual beam targets to produce [11C]carbon dioxide by the 14N(p,&#945;) 11C nuclear reaction.</li>
	<li>Approximately 20 min before unloading [11C]carbon dioxide from the cyclotron to the Module, start the validated time list for [11C]ER-176 synthesis by clicking the start button. Allow the CH4 column to cool down to 45 oC before receiving radioactive [11C]carbon dioxide (see Figure 3). Allow carbosphere column trap (CH4 trap) to cool to -75 oC.</li>
	<li>Let the method embedded in the Module for the conversion of [11C]carbon dioxide into [11C]methyl iodide, by way of a dry chemistry process, proceed as follows:
	<ol>
		<li>Convert [11C]carbon dioxide activity into [11C]methane by reacting with hydrogen gas at 350 oC over a nickel catalyst (Shimalite-Nickel). Use an ascarite trap (sodium hydroxide) to retain the unconverted [11C]carbon dioxide and formed water.</li>
		<li>Entrap the formed [11C]methane on a carbosphere column trap (CH4 trap) at -75 oC for further purification and concentration. To release the trapped [11C]methane, heat the carbosphere column to 80 oC.</li>
		<li>React the purified [11C]methane with elemental iodine at 720 oC to form [11C]methyl iodide ([11C]CH3I) by way of a helium recirculation gas pump where incident-generated hydrogen iodide is retained by another ascarite trap while unconverted [11C]methane returns into the circulation process.</li>
		<li>Entrap the formed [11C]CH3I at room temperature on the methyl iodide (MeI) column that has accumulated during the recirculation process.</li>
		<li>At the end of the circulation process, release the collected [11C]CH3I from the MeI trap by heating to 190 oC under helium flow (15 mL/min), by-passed from the MeTf column, and guided through a check-valve into the 1.5-mL stainless steel loop containing the preloaded precursor solution (step 1.5.1).</li>
	</ol>
	</li>
	<li>Once the [11C]CH3I has passed through the loop for 180 s, inject the reaction mixture onto the semi-prep HPLC column for purification. Use the following HPLC conditions: mobile phase of 37:63 (v/v) acetonitrile: 20 mM ammonium hydroxide at a flow rate of 5.0 mL/min and a UV wavelength of 235 nm; retention time (tR) of [11C]ER-176: approximately 12&#8211;14 min. See Figure 5.</li>
	<li>Collect the fraction sample into a large receiving flask containing 25 mL of sterile deionized water. Load the diluted mixture onto a C18 light solid phase extraction (SPE) cartridge.</li>
	<li>Wash product (1) with an additional 10 mL of sterile deionized water.</li>
	<li>Elute the desired product off the C18 light SPE with the use of 200-proof ethanol &#160;(1 mL). Direct this elution into a formulation flask preloaded with 0.9% sodium chloride for injection (6 mL).</li>
	<li>Further rinse the C18 light SPE with an additional 3 mL of 0.9% sodium chloride for injection via V4 reservoir.&#160;</li>
	<li>Collect the final solution in the formulation flask (~10 mL) and pass through a 0.22-&#956;m sterilizing filter into a preassembled sterile, apyrogenic, USP Type I 50 mL glass vial, sealed with a rubber septum and crimped with an aluminum cap.&#160;</li>
	<li>Using telemanipulators, remove an aliquot from the final product vial as a quality control (QC) sample. Subject this sample to numerous QC tests to ensure conformance with established&#160;specifications prior to release and administration to a patient (see Table 2). 
	NOTE: Apply this loop method procedure using [11C]CH3I for the radiolabeling of (2); generate and use [11C]CH3OTf to produce (3) and (4) (see Supplemental File 1).
	<ol>
		<li>To follow the typical quality control analysis conducted in the facility, use telemanipulators to transfer a sample from the final product vial into a TB syringe and then to the Quality Control room using a lead-shielded carrier.</li>
		<li>In a lead-shielded area (L-block), expel the sample into a pyrogen-free tube, and then dispense it into smaller glass vials (50&#8211;100 &#956;L) for HPLC and gas chromatography (GC) analyses.</li>
		<li>Dilute a second sample with pyrogen-free water to the appropriate concentration for bacterial endotoxin analysis.</li>
		<li>Inoculate a sample of the final product solution (in both tryptic soy broth (TSB) and fluid thioglycolate medium (FTM) media as a sterility sample). Incubate and observe these samples for growth over 14 days.</li>
		<li>Apply a small aliquot to a pH strip to visually determine the acidity/basicity of the final product solution.</li>
	</ol>
	</li>
</ol>
2.&#160;Reaction vessel method for the radiolabeling of [11C]ER-176 (1)
<ol>
	<li>Ensure the Module is thoroughly cleaned according to the internal cleaning protocol.
	<ol>
		<li>Use 1 N HCl, water, acetone, and acetonitrile for cleaning the reaction portion of the Module, and water followed by ethanol to clean and sterilize the formulation portion of the Module.</li>
		<li>Condition the methane (CH4) furnace at 350 oC for 20 min with a hydrogen gas flow of 100 mL/min prior to all productions performed on the Module. Cool to 45 oC before proceeding.</li>
		<li>Condition the CH4 trap at 120 oC for 20 min with a helium gas flow of 50 mL/min prior to all productions performed on the Module. Cool to 70 oC before proceeding.</li>
		<li>Condition the MeI trap at 190 oC for 20 min with a helium gas flow of 50 mL/min prior to all productions. Cool to 50 oC before proceeding. Ensure the V8 tubing is connected to the needle in the vessel.</li>
	</ol>
	</li>
	<li>Perform a leak test using helium gas, from the receiving valve (V24 to V16) to the internal MeI loop, and from V8 to the vessel using V23 as a source of seal/exhaust.</li>
	<li>Reagent loading for the Module:
	<ol>
		<li>Add the following: (R)-N-sec-butyl-4-(2-chlorophenyl)-quinazoline-2-carboxamide (1.0 &#177; 0.05 mg) in dimethyl sulfoxide-d6 (100 &#177; 10 &#956;L) and 6 N sodium hydroxide (4 &#956;L) to reactor vessel.</li>
		<li>Load the V2 reservoir with 1.0 mL of 37:63 acetonitrile:20 mM ammonium hydroxide.</li>
		<li>Load the V4 reservoir with 3.0 mL of 0.9% sodium chloride for injection.</li>
		<li>Load the V5 reservoir with 1.0 mL of 200-proof ethanol.</li>
		<li>Load the V6 reservoir with 10 mL of sterile deionized water.</li>
		<li>Fill the large receiving flask with 25 mL of sterile deionized water.</li>
		<li>Fill the formulation flask with 6.0 mL of 0.9% sodium chloride for injection.</li>
		<li>Ensure the delivery line is connected to the preassembled sterile final product vial.</li>
		<li>Fill up a reservoir with HPLC mobile phase (37:63 (v/v) acetonitrile:20 mM ammonium hydroxide) and connect to the HPLC pump. 
		NOTE: The semi-prep column used is the same as above and preconditioned with the mobile phase (4 column volumes).</li>
	</ol>
	</li>
	<li>During the preparation of the automated module, bombard carbon-11 cyclotron target (1% oxygen, 99% nitrogen) on an 11 MeV cyclotron at 55 &#956;A for 60&#8211;80 min on dual beam targets to produce [11C]carbon dioxide by the 14N(p,&#945;) 11C nuclear reaction.</li>
	<li>Approximately 20 min before unloading [11C]carbon dioxide from the cyclotron to the Module, start the validated time list for [11C]ER-176 synthesis by clicking the start button. Allow the CH4 column to cool down to 45 oC before receiving radioactive [11C]carbon dioxide (see Figure 3). Allow carbosphere column trap (CH4 trap) to cool to -75 oC</li>
	<li>Let the method embedded in the Module for the conversion of [11C]carbon dioxide into [11C]methyl iodide, by way of a dry chemistry process, proceed as follows:
	<ol>
		<li>Convert [11C]carbon dioxide activity into [11C]methane by reacting with hydrogen gas at 350 oC over a nickel catalyst (Shimalite-Nickel). Retain both unconverted [11C]carbon dioxide and formed water with an ascarite trap (sodium hydroxide).</li>
		<li>Entrap [11C]methane on a carbosphere column at -75 oC for further purification and concentration. To release the [11C]methane, heat the carbosphere column to 80 oC.&#160;</li>
		<li>React the purified [11C]methane with elemental iodine at 720 oC to form [11C]methyl iodide by way of a recirculation gas pump where incident-generated hydrogen is retained by another ascarite trap while unconverted [11C]methane returns into the circulation process, called internal loop recirculation.</li>
		<li>Entrap the formed [11C]methyl iodide at room temperature on the MeI column that has accumulated during the recirculation process.</li>
		<li>At the end of the circulation process, heat the porous polymeric adsorbent trap to 190 oC under helium flow (30 mL/min) to release the collected [11C]methyl iodide and guide it through a check-valve to the 3 mL reaction vessel for the carbon-11 radiolabeling of (1).</li>
	</ol>
	</li>
	<li>Once the generation and accumulation&#160;of&#160;[11C]methyl iodide have&#160;plateaued, by-pass the silver triflate column and direct the radioactive gas through V8 into the reaction vessel containing the precursor mixture. Allow the [11C]methyl iodide to bubble for 3 min, then seal the reaction vessel and heat it at 80 oC for 5 min.</li>
	<li>Once the labeling is complete, after 5 min, cool the reaction vessel to 30 oC and dilute with 1 mL of mobile phase from the V2 reservoir.</li>
	<li>Inject the mixture onto the semi-prep HPLC column for purification using the following HPLC conditions: mobile phase of 37:63 (v/v) acetonitrile: 20 mM ammonium hydroxide at a flow rate of 5.0 mL/min and a UV wavelength of 235 nm; tR of [11C]ER-176 (1): approximately 9&#8211;11 min, see Figure 6.</li>
	<li>Collect the fraction sample into the large receiving flask containing 25 mL of sterile deionized water. Load this diluted mixture onto a C18 light SPE cartridge.</li>
	<li>Wash the trapped product (1) with an additional 10 mL of sterile deionized water.</li>
	<li>Elute the desired product off the C18 light SPE with the use of 200 proof ethanol (1.0 mL), directed into a formulation flask preloaded with 0.9% sodium chloride for injection (6.0 mL).</li>
	<li>Rinse the C18 light SPE with an additional 3.0 mL of 0.9% sodium chloride for injection.&#160;</li>
	<li>Collect the final solution in the formulation flask (~10 mL). Pass this media through a 0.22-&#956;m sterilizing filter into a preassembled sterile, apyrogenic, USP Type I 50 mL glass vial, sealed with a rubber septum and crimped with an aluminum cap.&#160;</li>
	<li>Use telemanipulators to remove an aliquot from the final product vial as a quality control (QC) sample (see step 1.15). Subject the sample to several tests in which the radiotracer must pass all acceptance criteria prior to release and administration into a patient (see Table 2). 
	NOTE: Apply this reactor vessel method using [11C]CH3I for the radiolabeling of (2); generate and use [11C]CH3OTf to produce (3) and (4) (see Supplemental File 1).&#160;</li>
</ol>

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

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The authors declare they have no relevant or financial interests related to this research to disclose.

Traditional radiolabeling of terminal heteroatoms with carbon-11 iodomethane or methyl triflate involves bubbling the radioactive electrophilic gas into a reaction vessel, trapping it, and allowing the solution to react for extended periods of time18. The conventional bubbling of the heterogeneous reaction can be sluggish and may require heating to accelerate the reaction rate. Before purification, cooling the reaction media to room temperature may be necessary, followed by transferring to an HPLC loop with the use of solvent (e.g., mobile phase) for purification of the desired radiotracer. These steps are time-consuming, and fleeting radiolabeled products may be lost during the transfer of such materials.
At our facility, we have demonstrated the justification for using the loop method, where precursor solution is coated on an HPLC stainless steel loop, and the radiolabeling of the compound takes place inside the loop at room temperature. The loop is tied in-line with the delivery of the radioactive [11C]CH3I or [11C]CH3OTf and connected to the injection port of an HPLC system. No heating is required for the radiolabeling to occur, and for all cases shown in this manuscript, the reaction takes place in under 3 min.
The flow rate and size of the stainless steel HPLC loop appear to be critical for this process to work efficiently. Testing began by varying the flow rate of the carrier gas, helium, from 8.0 mL/min to 15.0 mL/min for the radioactive gas to be delivered to the loop. Buckley studied the importance of applying the correct flow rate as well as using the appropriate solvent and loop material15. For our system, the flow rate of 15 mL/min for either radioactive electrophilic species of [11C]CH3I or [11C]CH3OTf performs well for the radiolabeling of all four radiotracers discussed in this manuscript. The loop used for all syntheses is a stainless steel HPLC injection loop at 1.5 mL possessing an OD of 1/16 inch and an ID of 1 mm.
Comparing the two methods (reaction vessel versus loop method), in our hands, the loop method demonstrated increased radiolabeling efficiency along with a substantial increase in molar activity at end-of-synthesis (EOS) for the production of four human research-approved radiotracers. As an example, the radiotracer [11C]mHED&#160;(3)&#160;had a 1.6-fold increase in the isolated final product activity along with achieving double the molar activity at EOS. This trend of increased overall activity is observed across all four radiotracers (see&#160;Table 1). Additional favorable outcomes when using the loop method include a reduced setup time by 5 min and no need to clean the reaction vessel, saving the operator&#39;s time and use of solvents for a cleaning protocol.
Some drawbacks to this methodology include the limits on which radiotracers can be effectively labeled using the loop method. If heat is necessary for the radiolabeling, it is difficult to modify this system to allow for heating inside the HPLC loop. This system requires modifications to the plumbing; this feature of not having it ready &#39;out of the box&#39; may deter other users from performing such alterations to their automated platform14. Since supplemental plumbing and unions are required for this method (see&#160;Figure 4A-D), the potential for additional sites for radioactive releases increases when labeling under these conditions. It is prudent to perform a leak check prior to every run on the Module.
Our team has implemented the loop method used in the Investigational New Drug Application (IND) and Radioactive Drug Research Committee (RDRC)-approved production of four carbon-11 radiotracers. In our hands, this method proved to be a more efficient and higher-yielding process than the traditional reaction vessel method. Additional plumbing and adjustments to the flow rate of the carrier helium gas need to be considered when applying this method to most automated Modules. Finally, this method has limitations and is not suitable for certain carbon-11 radiotracers, such as [11C]UCB-J, which requires activation of the palladium (II) intermediate and heating24&#160;of the reaction mixture.

Among the modalities in molecular imaging, positron emission tomography (PET) is distinct in its manner of resolution of the biochemical processes associated with specific physiological targets or regions of interest1,2. The characteristic sensitivity and non-invasive nature of PET are harnessed for the&#160;in vivo&#160;visualization and quantification of disease pathophysiology, often revealing targets invisible by more anatomical imaging techniques such as computed tomography (CT)3&#160;or magnetic resonance imaging (MRI)4. Contemporary molecular imaging sees the combination of PET with CT or MRI (PET/CT or PET/MR, respectively), leveraging PET&#39;s high-contrast resolution and quantifiable imaging parameters to provide highly accurate attenuation correction maps (PET/CT) and improved spatial resolution (PET/MR)5, overcoming some hurdles presented by variability in the higher kinetic energies of positrons from radionuclides such as gallium-68 and rubidium-826. These dual-modality imaging techniques channel the hallmark attributes of each individual modality, furnishing clinicians or researchers with a wealth of co-registered anatomical and biochemical insights into the study subject5.
The clinical applicability of this imaging technique is vast, offering visualization and measurement of molecular-level physiological processes as diverse as glucose metabolism7,8, neurotransmitter receptor binding9, myocardial perfusion10, and various neurological conditions11. Outside clinical use, the inherent attributes of PET are aligned to play an integral role in diagnostic and therapeutic drug development, allowing for the quantification of parameters such as binding potential (BP), biodistribution, volume of distribution (VT), and drug-receptor occupancy (RO%) by direct observation of the interplay of pharmacology, pharmacokinetics, and pharmacodynamics. This in turn, contributes to determinations, including whether a compound reaches a target at an effective dose (ED50) concentration, the extent of effective penetration of the blood-brain barrier, the metabolic integrity of the compound, and appropriate dose and dosing interval11.
In developing a useful probe for PET imaging, upon the identification of an appropriate biomarker and selecting an associated ligand, radiolabeling of the biomolecule with a suitable PET radionuclide yields the radiotracer probe for the PET study. Among PET radionuclides for investigating biological, pharmacological, or medical questions, carbon-11 offers a combination of synthetic versatility and favorable physical characteristics that enables its widespread use across diverse biomolecules and eligible ligands6. With a 99.8% positron emission and 20.4-minute half-life12, carbon-11 allows repeated administration to subjects within short intervals while still permitting multi-step syntheses. However, these advantages necessitate a facility with on-site cyclotron and radiochemistry capabilities5.
Such facilities require reliable, powerful, and reproducible methylation methods that enable the radiolabeling of precursor molecules, often with the electrophilic [11C]iodomethane ([11C]CH3I) or [11C]methyl triflate ([11C]CH3OTf) substrate13. A radiosynthesis module, as supplied from the manufacturer, is typically configured for a reaction vessel approach to [11C]methylation reactions14. This involves cooling the vessel for effective [11C]iodomethane or [11C]methyl triflate retention upon delivery, sealing and heating the vessel to effectuate the reaction, quenching, and then transferring the reacted contents to a high-performance liquid chromatography (HPLC) system for semipreparative purification13. While effective15, this technique presents numerous potential structural points of failure, involving vial septa, needles, and associated transfer lines.
The need for a more reliable and reproducible methylation method guided the inquiry and pursuit of a captive solvent modification to many of our carbon-11 radioligand synthesis protocols. This approach aims to address the limitations of the conventional reaction vessel method while maintaining or improving radiolabeling efficiency.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67406/67406fig2.jpg" /> 
Figure 2: Design and flow of the reaction vessel synthesis and loop method. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Captive solvent chemistry offers the promise of efficient trapping of, and reaction with, the radiolabeling reagent by means of spreading of the precursor solution over a large area of a supporting material or structure, and then directing the gaseous labeling reagent into contact with the coated material16,17. This enhances the extent and quality of contact between the two reactant phases16,18. Numerous implementations and variations of this technique have been documented as early as 198516,17,18,19,20, and it has found application with [11C]methyl iodide, [11C]methyl triflate, as well as Grignard reagent [11C]carbon dioxide radiolabeling reactions20. Further refinement was presented by the discussion of captive solvent &#34;loop method&#34; chemistry, originally described by Wilson et al., which does not require any additional supports to that already offered by the HPLC purification loop, nor heating or cooling of the reaction environment13. Captive Solvent &#34;loop method&#34; radiolabeling was found to impart iodomethane and methyl triflate [11C]methylation reactions with the virtues of minimal transfer loss, high radiochemical yield, high molar activity, decreased reaction time, and simplicity7,21,22,23.
Herein we describe our group&#39;s implementation of the &#34;loop method&#34; [11C]methylation technique originally described by Wilson13, and others thereafter14,15,18,21,22,23, (see&#160;Figure 2) by means of mechanical modification (see&#160;Figure 3) to our synthesis module (hereafter referred to as the&#160;Module). Hewing as closely to the aspirational ethos of simplicity as possible, these mechanical modifications were minimal and accessible, resulting in an overall reduction of engineering complexity, and only the addition of essential components to those already installed by the module&#39;s manufacturer as associated with a default reaction vessel radiolabeling arrangement. This is reflected in the decision to utilize the stainless steel HPLC purification loop already supplied and preinstalled by the module manufacturer, as described below, which proved compatible and effective with the syntheses examined. We discuss, in full detail, the complete and validated loop method radiolabeling protocol utilized in the clinical research production for the synthesis of the radiotracer [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide,[11C]ER-176&#160;(1), using carbon-11 iodomethane. Further, we compare numerous attributes related to radiolabeling efficiency as conducted by both the reactor method and the loop method across three additional radioligands, which include (S,S)-[11C]methylreboxetine ([11C]MRB&#160;(2)),[11C]-meta-hydroxyephedrine ([11C]mHED&#160;(3)), and 2-[4-[(11C)methylamino]phenyl]-1,3-benzothiazol-6-ol ([11C]PiB&#160;(4)) developed in our facility, as determined by analysis of synthesized batches (see&#160;Table 1&#160;and&#160;Figure 1). This comparison illustrates the clear boon to parameters such as radiochemical yield and molar activity associated with the implementation of &#34;loop method&#34; radiolabeling, afforded by accessible and simple module modifications of minimal cost to the radiochemistry laboratory.

<table><tbody><tr><td>2-(4&#039;-Aminophenyl)-6-hydroxybenzothiazole</td><td>ABX</td><td>5101</td><td>Precursor for PiB synthesis (6-OH-BTA-0)</td></tr><tr><td>(2S,3S)-Desethylreboxetine</td><td>ABX</td><td>4407</td><td>Precursor for MRB synthesis</td></tr><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>695092</td><td>Reagent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Acetone-d&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>444863</td><td>Solvent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Acetonitrile, HPLC-grade</td><td>Sigma Aldrich</td><td>34998</td><td>Various concentrations used in the mobile phase for radiotracer productions</td></tr><tr><td>Acetonitrile-d&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>151807</td><td>Solvent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]mHED</td></tr><tr><td>Ammonium formate</td><td>Sigma Aldrich</td><td>798568</td><td>Reagent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]MRB</td></tr><tr><td>Ammonium hydroxide</td><td>Ricca Chemical</td><td>642-16</td><td>Reagent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]ER-176</td></tr><tr><td>Analytical balance</td><td>Mettler Toledo&amp;nbsp;</td><td>M-XS104</td><td>Balance used to weigh out materials for productions</td></tr><tr><td>Ascarite II</td><td>Thermo Fisher</td><td>CN-C049U90</td><td>Used in drying columns for FxC Pro Module</td></tr><tr><td>Biosafety cabinet</td><td>Comecer&amp;nbsp;</td><td>M-BH4</td><td>Used for FPV assembly</td></tr><tr><td>(R)-N-sec-Butyl-4-(2-chlorophenyl)-quinazoline-2-carboxamide</td><td>ABX</td><td>1665.0001</td><td>ER-176 precursor</td></tr><tr><td>C18 Light Sep-Pak cartridge</td><td>WATERS</td><td>WAT023501</td><td>Solid phase extraction cartridge used in the synthesis of carbon-11 radiotracers</td></tr><tr><td>Carboxen, 60 &amp;ndash; 80 mesh</td><td>Supelco</td><td>CN-10478-U</td><td>Used in the FxC Pro Module</td></tr><tr><td>Compressed &amp;ndash; NOS (99% nitrogen/1% oxygen)</td><td>Airgas&amp;nbsp;</td><td>CN-X02NI99C3003091</td><td>Target gas used in cyclotron bombardment</td></tr><tr><td>Dispensing hot cell (DHC)</td><td>Comecer&amp;nbsp;</td><td>M-MIP1-1390</td><td>Dispensing radiotracers</td></tr><tr><td>Dose calibrator&amp;nbsp;</td><td>Capintec</td><td>M-CRC-55t</td><td>Measuring activity of radiotracers</td></tr><tr><td>Endosafe nexgen-PTS</td><td>Charles River&amp;nbsp;</td><td>PTS150</td><td>Endosafe PTS, portable test sytem</td></tr><tr><td>Endotoxin PTS - Limulus Amebocyte Lysate (LAL) Test Cartridge</td><td>Charles River&amp;nbsp;</td><td>PTS20F</td><td>LAL cartridges used to test endotin levels on radiotracers</td></tr><tr><td>Ethanol, 200 proof, HPLC-grade</td><td>Sigma Aldrich</td><td>459828</td><td>Used for&amp;nbsp; final product and&amp;nbsp; mobile phase of ER, MRB, mHED and PiB</td></tr><tr><td>Gas Chromatogram 2030</td><td>Shimadzu</td><td>M-GC-2030</td><td>Measure excipients from radiotracer productions</td></tr><tr><td>Graphpac</td><td>Supelco, Millipore Sigma</td><td>10258</td><td>Used in FxC Pro Module for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]ER-176, [&lt;sup&gt;11&lt;/sup&gt;C]MRB, [&lt;sup&gt;11&lt;/sup&gt;C]mHED and [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Helium, research grade</td><td>Airgas&amp;nbsp;</td><td>CN-HER-300</td><td>Helium tank, 99.9999% (research grade tank)</td></tr><tr><td>Shimadzu LC-20 Series&amp;nbsp;</td><td>Shimadzu</td><td>Various&amp;nbsp;</td><td>Analytical HPLC system</td></tr><tr><td>Hydrogen, ultra high purity grade&amp;nbsp;&amp;nbsp;</td><td>Airgas&amp;nbsp;</td><td>CN-HYUHP-300</td><td>Hydrogen tank used for the FxC Pro Module</td></tr><tr><td>Iodine</td><td>Thermo Fisher</td><td>I35</td><td>Used in FxC Pro Module, conversion of [&lt;sup&gt;11&lt;/sup&gt;C]CH&lt;sub&gt;4&lt;/sub&gt; to [&lt;sup&gt;11&lt;/sup&gt;C]CH&lt;sub&gt;3&lt;/sub&gt;I</td></tr><tr><td>Luna 10 mm C18 (2) 100 &amp;Aring; 250 x 10 mm column</td><td>Phenomenex</td><td>00G-4253-N0</td><td>Semi-prep column for MRB synthesis</td></tr><tr><td>Luna 5 mm C18 (2) 100 &amp;Aring; 250 x 10 mm column</td><td>Phenomenex</td><td>00G-4252-N0</td><td>Semi-prep column for PiB synthesis</td></tr><tr><td>Macherey-Nagel MN VP 250/10 Nucleosil 100-5 C18 Nautilus column</td><td>Macherey-Nagel</td><td>715412.1</td><td>Semi-prep column for mHED synthesis</td></tr><tr><td>Metaraminol (free base)</td><td>ABX</td><td>3380.0001</td><td>Precursor used in the production of [&lt;sup&gt;11&lt;/sup&gt;C]mHED</td></tr><tr><td>Milli-Q Direct 8 DI system</td><td>Millipore</td><td>M-ZROQ00800</td><td>De-ionized water system</td></tr><tr><td>N,N-Dimethylformamide-d&lt;sub&gt;7&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>189979</td><td>Solvent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]MRB</td></tr><tr><td>Needle, 18 G x 1</td><td>Becton Dickinson</td><td>BD 305195</td><td>Needles used at various stages of production setup</td></tr><tr><td>Needle, 20 G x 1-1/2&amp;rdquo;</td><td>Becton Dickinson</td><td>BD 305176</td><td>Needles used at various stages of production setup</td></tr><tr><td>Needle, 22 G x 4&amp;rdquo;, Spinal&amp;nbsp;</td><td>Air-Tite Products</td><td>N224</td><td>Needles used at various stages of production setup</td></tr><tr><td>Onyx Monolithic C18 100 x 10 mm column</td><td>Phenomenex</td><td>CH0-7878</td><td>Semi-prep column for ER-176 synthesis</td></tr><tr><td>Phosphorus Pentoxide with Sicapent indicator</td><td>Sigma Aldrich</td><td>79610</td><td>Used in the synthesis Modules for carbon-11 radiotracer productions</td></tr><tr><td>Porapak N</td><td>Waters</td><td>WAT027047</td><td>Material used to pack MeI column in FxC Pro Module</td></tr><tr><td>Pressure gauge</td><td>Omega</td><td>M-DPG1100B-100G</td><td>Used for filter integrity test</td></tr><tr><td>Shimalite-Ni</td><td>Shimadzu Corporation</td><td>221-66062</td><td>Material packed in CH&lt;sub&gt;4&lt;/sub&gt; oven of FxCPro Module for conversion from [&lt;sup&gt;11&lt;/sup&gt;C]CO&lt;sub&gt;2&lt;/sub&gt; to [&lt;sup&gt;11&lt;/sup&gt;C]CH&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Siemens Eclipse 11-MeV cyclotron</td><td>Siemens</td><td>RDS 111</td><td>Particle accelerator used to generate [&lt;sup&gt;11&lt;/sup&gt;C]CO&lt;sub&gt;2&lt;/sub&gt; gas for carbon-11 productions</td></tr><tr><td>Silver Triflate</td><td>Sigma Aldrich</td><td>483346</td><td>Material packed in triflate column of the FxC Pro Module for the production of [&lt;sup&gt;11&lt;/sup&gt;C]mHED and [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Sodium chloride injection (saline, 0.9%), 50 mL</td><td>Hospira</td><td>0409-4888-50</td><td>Saline used in the formulation process of radiotracers</td></tr><tr><td>Sodium chloride injection (saline, 0.9% ), 500 mL</td><td>Braun</td><td>L8001</td><td>Reagent used in the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]mHED, and mobile phase.</td></tr><tr><td>Sodium hydroxide</td><td>Sigma Aldrich</td><td>221465</td><td>Reagent used in the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]ER-176 and other components at different concentration.</td></tr><tr><td>Sodium iodide detector</td><td>Eckert &amp;amp; Ziegler</td><td>PMT/Na-BFC3200</td><td>Gamma detector used in-line with HPLC unit</td></tr><tr><td>Sterile vial, 50 mL</td><td>ALK Allergy</td><td>SEV50</td><td>50 mL sterile vials used as the&amp;nbsp;</td></tr><tr><td>Stainless steel loop</td><td>GE</td><td>980314/IEG-005118</td><td>HPLC injection loop at 1.5 mL possessing an OD 1/16&amp;rdquo;, ID: 1 mm.</td></tr><tr><td>Syringe, 1 mL</td><td>Braun</td><td>NJ-9166017-02</td><td>Syringe used at various stages of production set-up</td></tr><tr><td>Syringe, 10 mL</td><td>Henke Sass Wolf</td><td>4100-X00V0</td><td>Syringe used at various stages of production set-up</td></tr><tr><td>Syringe, 20 mL</td><td>Henke Sass Wolf</td><td>4200-X00V0</td><td>Syringe used at various stages of production set-up</td></tr><tr><td>Syringe, 5 mL</td><td>Becton Dickinson</td><td>309632</td><td>Syringe used at various stages of production set-up</td></tr><tr><td>TracerLab FX2C Module&amp;nbsp;</td><td>GE</td><td>M-P5360QB</td><td>Automated module of production of carbon-11 radiotracers</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td>Reagent used for the synthesis of [&lt;sup&gt;11&lt;/sup&gt;C]PiB</td></tr><tr><td>Tuberculin Syringe, with 21 G x 1 Needle, Single-Use, Sterile (1 mL)</td><td>Becton Dickinson</td><td>BD 309624</td><td>Syringe used in pre-assembled final product vials</td></tr></tbody></table>

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.

The New York University Langone Health (NYULH) Radiochemistry group provides various carbon-11, fluorine-18, and gallium-68 radiotracers used for both human research and preclinical applications. Several methods are utilized for the production of the PET radiotracers. Our team employs the loop method for the synthesis of (1), (2), (3), and (4) (see&#160;Figure 1&#160;and&#160;Figure 8). After production is complete, an aliquot is removed from the sterile final product vial. This sample is used for both the inoculation of the final product solution (in both tryptic soy broth (TSB) and fluid thioglycolate medium (FTM) media as a sterility sample) as well as a representative sample of the bulk solution for QC tests. Each radiotracer is subjected to QC tests before the product is released for administration (see Table 2).
Quality control testing includes the visualization of product appearance, verification of filter integrity, determination of radionuclidic identity, pH, radiochemical identity (radio-HPLC), radiochemical purity (radio-HPLC), chemical purity (HPLC), molar activity, strength, residual solvent, endotoxins, and sterility (see Table 2). The following results were obtained from the clinical production of each above-mentioned radiotracer (see Table 1).
For a representative analytical HPLC chromatogram, see&#160;Figure 7&#160;and Supplemental File 1. Every radiotracer is required to pass all quality control specifications (see Table 2) before they can be released and administered to a subject.
Refer to the Supplemental File 1 for amounts of precursor and reagents as well as the analytical HPLC chromatograms of [11C]MRB (2) (Supplemental&#160;Figure 1), [11C]mHED (3) (Supplemental&#160;Figure 2), and [11C]PiB (4) (Supplemental&#160;Figure 3).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Compound</td>
			<td>Parameters</td>
			<td>Loop Method&#160; (avg &#177; std)</td>
			<td>Reactor Method&#160; (avg &#177; std)</td>
		</tr>
		<tr>
			<td rowspan="7">[11C]ER-176</td>
			<td>number of productions</td>
			<td>25</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Start of Synthesis</td>
			<td>86 &#177; 5.0 GBq</td>
			<td>52 &#177; 25.7 GBq</td>
		</tr>
		<tr>
			<td>End of Synthesis</td>
			<td>5.4 &#177; 2.2 GBq</td>
			<td>1.6 &#177; 0.5 GBq</td>
		</tr>
		<tr>
			<td>Radiochemical Purity</td>
			<td>99.6 &#177; 0.6%</td>
			<td>99.9 &#177; 0.1%</td>
		</tr>
		<tr>
			<td>ER-176 concentration</td>
			<td>1.1 &#177; 0.5 &#956;g/mL</td>
			<td>0.63 &#177; 0.37 &#956;g/mL</td>
		</tr>
		<tr>
			<td>molar activity</td>
			<td>194 &#177; 66 GBq/&#956;mol</td>
			<td>132 &#177; 78 GBq/&#956;mol</td>
		</tr>
		<tr>
			<td>Total Synthesis Time</td>
			<td>36 &#177; 3 min</td>
			<td>44 &#177; 6 min</td>
		</tr>
		<tr>
			<td rowspan="7">[11C]MRB</td>
			<td>number of productions</td>
			<td>70</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Start of Synthesis</td>
			<td>84 &#177; 5.4 GBq</td>
			<td>39 &#177; 11.9 GBq</td>
		</tr>
		<tr>
			<td>End of Synthesis</td>
			<td>3.0 &#177; 1.2 GBq</td>
			<td>1.9 &#177; 0.7 GBq</td>
		</tr>
		<tr>
			<td>Radiochemical Purity</td>
			<td>99.5 &#177; 0.5%</td>
			<td>99.7 &#177; 0.8%</td>
		</tr>
		<tr>
			<td>MRB concentration</td>
			<td>0.52 &#177; 0.24 &#956;g/mL</td>
			<td>0.68 &#177; 0.41 &#956;g/mL</td>
		</tr>
		<tr>
			<td>molar activity</td>
			<td>190 &#177; 50 GBq/&#956;mol</td>
			<td>99 &#177; 55 GBq/&#956;mol</td>
		</tr>
		<tr>
			<td>Total Synthesis Time</td>
			<td>35 &#177; 3 min</td>
			<td>42 &#177; 3 min</td>
		</tr>
		<tr>
			<td rowspan="7">[11C]mHED</td>
			<td>number of productions</td>
			<td>5</td>
			<td>11</td>
		</tr>
		<tr>
			<td>Start of Synthesis</td>
			<td>69 &#177; 10.5 GBq</td>
			<td>82 &#177; 4.3 GBq</td>
		</tr>
		<tr>
			<td>End of Synthesis</td>
			<td>5.5 &#177; 1.3 GBq</td>
			<td>3.3 &#177; 1.0 GBq</td>
		</tr>
		<tr>
			<td>Radiochemical Purity</td>
			<td>98.2 &#177; 1.3%</td>
			<td>99.1 &#177; 0.7%</td>
		</tr>
		<tr>
			<td>mHED concentration</td>
			<td>0.40 &#177; 0.10 &#956;g/mL</td>
			<td>0.52 &#177; 0.37 &#956;g/mL</td>
		</tr>
		<tr>
			<td>molar activity</td>
			<td>301 &#177; 48 GBq/&#956;mol</td>
			<td>155 &#177; 77 GBq/&#956;mol</td>
		</tr>
		<tr>
			<td>Total Synthesis Time</td>
			<td>27 &#177; 4 min</td>
			<td>32 &#177; 2 min</td>
		</tr>
		<tr>
			<td rowspan="7">[11C]PiB</td>
			<td>number of productions</td>
			<td>51</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Start of Synthesis</td>
			<td>86 &#177; 5.4 GBq</td>
			<td>57 &#177; 17.2 GBq</td>
		</tr>
		<tr>
			<td>End of Synthesis</td>
			<td>3.2 &#177; 0.8 GBq</td>
			<td>1.4 &#177; 0.2 GBq</td>
		</tr>
		<tr>
			<td>Radiochemical Purity</td>
			<td>97.0 &#177; 1.5%</td>
			<td>99.1 &#177; 1.4%</td>
		</tr>
		<tr>
			<td>PiB concentration</td>
			<td>0.22 &#177; 0.51 &#956;g/mL</td>
			<td>0.30 &#177; 0.24 &#956;g/mL</td>
		</tr>
		<tr>
			<td>molar activity</td>
			<td>201 &#177; 68 GBq/&#956;mol</td>
			<td>207 &#177; 124 GBq/&#956;mol</td>
		</tr>
		<tr>
			<td>Total Synthesis Time</td>
			<td>35 &#177; 2 min</td>
			<td>36 &#177; 5 min</td>
		</tr>
	</tbody>
</table>
Table 1: Results from the production of [11C]ER-176 (1), [11C]MRB (2), [11C]&#160;m&#160;HED (3), and[11C]PiB (4) by way of loop method or reaction vessel method. All values are reported at end-of-synthesis. Abbreviations: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide; [11C]MRB = (S,S)-[11C]methylreboxetine; [11C]mHED = [11C]-meta-hydroxyephedrine; [11C]PiB = 2-[4-[(11C)methylamino]phenyl]-1,3-benzothiazol-6-ol.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67406/67406fig1.jpg" /> 
Figure 1: Structures of [11C]ER-176 (1), [11C]MRB (2), [11C]&#160;m&#160;HED (3), and [11C]PiB (4). Abbreviations: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide; [11C]MRB = (S,S)-[11C]methylreboxetine; [11C]mHED = [11C]-meta-hydroxyephedrine; [11C]PiB = 2-[4-[(11C)methylamino]phenyl]-1,3-benzothiazol-6-ol. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig1large.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/67406/67406fig3.jpg" /> 
Figure 3: Modifications made to the automated Module. In red represents the replumbing of the synthesis module to incorporate the loop for the production of PET radiotracers by way of carbon-11 methylation. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig3large.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/67406/67406fig4.jpg" /> 
Figure 4: Module plumbing and re-plumbing to incorporate the loop method for carbon-11 methylation of PET radiotracers. (A) HPLC injection loop with union connectors. (B) Reaction vessel method. The red arrow showing the V8 connection. (C) Loop method re-plumbing, by-passing the reactor vessel. Note that the V8 connection to the HPLC inlet bypasses the reaction vessel, allowing [11C]iodomethane or [11C]methyl triflate direct access to the HPLC loop. (D) Placement of the union in regards to reactor vessel. Note the connection to the stainless steel HPLC loop. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig4large.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/67406/67406fig5.jpg" /> 
Figure 5: Semi-prep HPLC profiles of ER-176 and [11C]ER-176 with loop method production. (A) Semi-prep HPLC profile for ER-176 (1) by way of loop method production at UV = 235 nm; tR&#160;= 13.2 min. (B) Semi-prep radio-HPLC profile for [11C]ER-176 (1); tR&#160;= 12.4 min. Conditions: mobile phase of 37:63 (v/v) acetonitrile: 20 mM ammonium hydroxide at a flow rate of 5.0 mL/min. Abbreviation: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67406/67406fig6.jpg" /> 
Figure 6: Semi-prep HPLC profiles of ER-176 and [11C]ER-176 with reactor method production. (A) Semi-prep HPLC profile for ER-176 (1) by way of reactor method production at UV = 235 nm; tR&#160;= 9.8 min. (B) Semi-prep radio-HPLC profile for [11C]ER-176 (1); tR&#160;= 9.2 min. Conditions: mobile phase of 37:63 (v/v) acetonitrile: 20 mM ammonium hydroxide at a flow rate of 5.5 mL/min. Abbreviations: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide; tR&#160;= retention time. <a href="https://www.jove.com/files/ftp_upload/67406/67406fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/67406/67406fig7.jpg" /> 
Figure 7: Analytical HPLC profiles for ER-176. (A) Analytical HPLC profile of UV spectra for ER-176 (1) at 235 nm; tR&#160;= 6.10 min. (B) Analytical HPLC profile of radiotracer for [11C]ER-176 (1); tR&#160;= 6.36 min. Conditions: 10 &#181;m C18 (2) 100 <img alt="Equation 1" src="/files/ftp_upload/67406/67406eq1.jpg" style="font-size: 14px;" />&#160;LC Column 250 x 4.6 mm; methanol/water 74/26 with a flow rate of 1.5 mL/min. Abbreviation: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide; tR&#160;= retention time <a href="https://www.jove.com/files/ftp_upload/67406/67406fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/67406/67406fig8.jpg" /> 
Figure 8: Synthetic Scheme for Synthesis of Carbon-11 Compounds. (i) The formation of [11C]CH3I and [11C]CH3OTf, and (iia.) the radiolabeling of [11C]ER-176 (1), (iib.) [11C]MRB (2), (iic.)[11C]mHED (3), and (iid.) [11C]PiB (4). <a href="https://www.jove.com/files/ftp_upload/67406/67406fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Test/Parameters</td>
			<td>Specifications</td>
		</tr>
		<tr>
			<td>Appearance (visual inspection)</td>
			<td>Clear, colorless solution free from particulate matter</td>
		</tr>
		<tr>
			<td>Membrane filter integrity</td>
			<td>&#8805; 50 psi</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>4.5 - 8.0</td>
		</tr>
		<tr>
			<td>Radionuclidic identity (half-life)</td>
			<td>19.3 - 21.3 min</td>
		</tr>
		<tr>
			<td>Radiochemical identity (radio-HPLC)</td>
			<td>0.90 &#8804; tR Prod / t R Std &#8804; 1.10</td>
		</tr>
		<tr>
			<td>Radiochemical purity (radio-HPLC)</td>
			<td>&#8805; 95.0%&#8224;</td>
		</tr>
		<tr>
			<td>Drug concentration (HPLC)</td>
			<td>see notes*</td>
		</tr>
		<tr>
			<td>Total chemical impurities (HPLC)</td>
			<td>see notes**</td>
		</tr>
		<tr>
			<td>Molar activity (@ EOS)</td>
			<td>&#62; 9.25 GBq/&#956;mol</td>
		</tr>
		<tr>
			<td>Residual acetonitrile (GC)</td>
			<td>&#8804; 410 ppm</td>
		</tr>
		<tr>
			<td>Residual acetone (GC)</td>
			<td>&#8804; 5,000 ppm</td>
		</tr>
		<tr>
			<td>Residual dimethyl sulfoxide (GC)</td>
			<td>&#8804; 5,000 ppm</td>
		</tr>
		<tr>
			<td>Residual ethanol (GC)</td>
			<td>&#8804; 10% (v/v)</td>
		</tr>
		<tr>
			<td>Residual methanol (GC)</td>
			<td>&#8804; 3,000 ppm</td>
		</tr>
		<tr>
			<td>Residual N,N-dimethylformamide (GC)</td>
			<td>&#8804; 880 ppm</td>
		</tr>
		<tr>
			<td>Limulus Amoebocyte Lysate (LAL)</td>
			<td>&#8804; 17.5 EU/mL</td>
		</tr>
		<tr>
			<td>Sterility (initiate within 30 h)</td>
			<td>Sterile (14 days)</td>
		</tr>
		<tr>
			<td>Preparation records</td>
			<td>Complete and accurate</td>
		</tr>
		<tr>
			<td>Labels</td>
			<td>Complete, accurate, reconciled</td>
		</tr>
	</tbody>
</table>
Table 2: Quality control criteria for the approval or rejection of carbon-11 radiotracers. &#8224; Radiochemical purity &#8805; 90.0% for [11C]mHED; * Drug Concentration: [11C]ER-176 &#8804; 10 &#181;g/dose; [11C]MRB &#8804; 10 &#181;g/dose; [11C]mHED &#8804; 50 &#181;g/dose; [11C]PiB &#8804; 13.4 &#181;g/dose; ** Total chemical impurities: [11C]ER-176 &#8804; 1.0 &#181;g/dose; [11C]MRB &#8804; 1.0 &#181;g/dose; [11C]mHED &#8804; 5.0 &#181;g/dose; [11C]PiB &#8804; 1.34 &#181;g/dose. Abbreviations: [11C]ER-176 = [11C]-(R)-N-sec-butyl-4-(2-chlorophenyl)-N-methylquinazoline-2-carboxamide; [11C]MRB = (S,S)-[11C]methylreboxetine; [11C]mHED = [11C]-meta-hydroxyephedrine; [11C]PiB = 2-[4-[(11C)methylamino]phenyl]-1,3-benzothiazol-6-ol; EOS = end of synthesis; GC = gas chromatography; LAL = Limulus Amebocyte Lysate.
Supplemental File 1: Chemicals and materials, reagents used for loop or reactor vessel method, Quality Control testing, analytical HPLC chromatograms. <a href="https://www.jove.com/files/ftp_upload/67406/67406_JoVe_Supplementary File_Final_CarberryEdit.zip" target="_blank">Please click here to download this file.</a>

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

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, ...

enhancing-efficiency-radiolabeling-yields-carbon-11-radioligands-for

Universidad San Sebastian

Research

JoVE Journal

Chemistry

1.2K Views.  New York University Grossman School of Medicine. This protocol presents a detailed procedure to prepare four carbon-11 radiotracers by way of the “loop method” [11C]methylation. We describe the procedure to synthesize [11C]ER-176,  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 [11

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

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

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 ...

Cancer Research

Optimization of Radiochemical Reactions using Droplet Arrays

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction optimization or novel radiopharmaceutical development since each data point involves significant reagent consumption, and contamination of the apparatus requires time for radioactive decay before the next use. To address these limitations, a platform for performing arrays of miniature droplet-based reactions in parallel, each confined within a surface-tension trap on a patterned polytetrafluoroethylene-coated silicon "chip", was developed. These chips enable rapid and convenient studies of reaction parameters including reagent concentrations, reaction solvent, reaction temperature and time. This platform permits the completion of hundreds of reactions in a few days with minimal reagent consumption, instead of taking months using a conventional radiosynthesizer.

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

Current automated radiosynthesizers are designed to produce large clinical batches of radiopharmaceuticals. They are not well suited for reaction ...

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 ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Immunology and Infection

Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs that mimic the structure of natural biomolecules. Cells utilize analogs in their endogenous biochemical processes, producing compounds that are labeled. The label allows for the incorporation of detection and affinity tags, which can then be used to elucidate metabolic pathways using other biochemical analytical techniques, such as SDS-PAGE and NMR.
This video introduces the concepts of metabolic labeling and show two general procedures.&#160; The first uses isotopic-labeling, to characterize the phosphorylation of a protein. The second covers a photoreactive labeling to characterize protein-protein interaction within a Also three applications of metabolic labeling are presented: labeling plant material, labeling RNA to measure kinetics and labeling glycans in developing embryos.

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Metabolic labeling is used to investigate the machinery of a cell. This is accomplished using chemical analogs to probe the biochemical transformations and modifications that occur. This video will show the principles of metabolic labeling, typical isotopic and photoreactive labeling procedures, and some applications.
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Metabolic labeling can be conducted using a number of strategies. Here we will describe isotopic, photoreactive, and bio-orthogonal labeling.
Isotopic labeling is performed using structural analogs that are chemically identical to their natural counterparts, but have uncommon isotopes incorporated into their structure. In this L-lysine analog the carbon and nitrogen atoms are replaced with carbon-13 and nitrogen-15. Cells cultured in the presence of isotopic analogs will incorporate them into their biochemical structures. Metabolites are collected from the cells and purified for analysis. Samples with stable isotopes are analyzed using techniques such as mass spectrometry or NMR spectroscopy. Samples with radioactive labels are analyzed using liquid scintillation counting and x-ray films, which will be demonstrated in the isotopic labeling protocol.
Photoreactive labels are functional groups incorporated into proteins, which are stable until exposed to ultraviolet light. The functional group forms a reactive radical, which binds to the nearest protein. A common example, L-photo-leucine, contains a diazirine ring, which is a photoreactive crosslinker. In contrast to isotopic labeling, there is some chemical dissimilarity between photo-reactive chemical analogs and their natural counterparts. Cells may preferentially incorporate natural compounds over analogs. Therefore, it is important to perform photo-reactive labeling in medium free of the compound being mimicked. Once exposed to ultraviolet radiation, the photo-reactive groups in a labeled protein become unstable and highly reactive, causing it to cross-link with interacting proteins, creating a protein complex. Cross-linked complexes, act as snapshots that can then be analyzed using SDS-PAGE and mass spectrometry methods. This provides insights into what reactions are occurring in the metabolic pathway by identifying reaction species and how they interact by determining binding sites.
Bioorthogonal labeling strategies utilize analogs with small functional groups that have little to no reactivity with natural biomolecules. For example, azides are small functional groups, whose reactivity is said to be orthogonal to biochemical reactions. In the Staudinger ligation, a phosphine group attacks the azido group. This yields a transition state that intramolecularly reacts with a nearby ester, resulting in an amine-bonded ligand. Bioorthogonal functional groups incorporated into biomolecules can be ligated with detection tags such as fluorescent functional groups, and affinity tags such as antigens.
Now that some concepts and strategies for metabolic labeling have been discussed, let&#39;s look at the process in the laboratory.
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The first step in a metabolic labeling experiment is to collect the protein of interest. To do this, cells are grown on a plate, and an expression method is used to promote synthesis of the desired protein. In this example, leucine-rich repeat kinases, or LRRK, are expressed. Disodium phosphate, containing radioactive phosphorus-32, is used as the analog. Proper measures must be taken to protect against ionizing radiation. This includes setting up a work area, wearing proper protective equipment, and checking for radioactive contamination. Once safety measures have been taken, the medium containing the isotopic analogs is prepared. The medium from the culture is removed and, replaced with one containing the isotopic chemical analogs and then incubated. Following incubation, the cells are lysed. The lysate is collected and purified.
After purification, proteins are resolved using SDS-PAGE and then transferred to a PVDF membrane. Autoradiography is performed by exposing the membrane to x-ray film and measured using a phosphor imager. Western blotting is used to measure relative protein levels in the PVDF membrane. In this example the phosphorylation levels of leucine-rich repeat kinases synthesized in 293T cells were measured. The autoradiogram shows how much phosphorous was incorporated into the protein. Western blotting elucidates the levels of the LRRK proteins. Image analysis software is used to obtain quantitative data of phosphorylation levels of the proteins.
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In this next procedure, photoreactive labeling is demonstrated. First, the cells are prepared and cultured. The photoreactive analog is added to the cells at the mid-log phase and incubated. In this procedure p-benzoylphenylalanine is used. Samples are collected over intervals and put on ice. The samples are then exposed to obtain snapshots of the biochemical pathways over time. The proteins of interest are then purified and resolved using SDS-PAGE.
A photoreactive labeling strategy was used to identify compounds that interact with the protein of interest. Immunodetection with Western blotting shows protein bands that indicate higher molecular weight proteins are present in the irradiated samples. These are from cross-linking due to protein-protein interaction occurring during the UV irradiation.
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Now that we&#39;ve reviewed metabolic labeling procedures, let&#39;s look at some of the ways the process is used.
Metabolic labeling concepts can be extended to multicellular organisms. Plants are grown in a sealed environment, rich in stable isotopes to produced labeled plant material. Carbon dioxide containing carbon-13 is added to the enclosure, while nitrogen-15 rich fertilizer is used. The resulting harvested plant material can help answer questions about carbon and nitrogen cycling from the ecosystem.
Labeling enables the separation of newly synthesized RNA from older RNA. By changing the initial concentration of analog, the kinetics of new RNA synthesis can be determined. The results show that the concentration of 4-thiouridine affects how much new RNA is transcribed. Additionally, incorporation rates of the label into RNA can be directly quantified with a spectrophotometer.
Using biorthogonal click chemistry, glycans in a zebra fish embryo can be labeled. The eggs are injected with a labeling compound that results in alkyne labels on the glycans. The glycans in the larvae are then ligated to a dye compound at the desired development stage. The glycans in the embryos are then imaged. Glycans produced at different time points can be identified by labeling using different colors at different stages of embryo development.
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You&#39;ve just watched JoVE&#39;s video on metabolic labeling. This video described the concepts behind metabolic labeling and their strategies, went over two general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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Isotopic, Photoreactive and Bioorthogonal Metabolic Labeling

Metabolic labeling is used to probe the biochemical transformations and modifications that occur in a cell. This is accomplished by using chemical analogs ...

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