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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(4&#39;-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 [11C]PiB</td>
		</tr>
		<tr>
			<td>Acetone-d6</td>
			<td>Sigma Aldrich</td>
			<td>444863</td>
			<td>Solvent used for the synthesis of [11C]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-d3</td>
			<td>Sigma Aldrich</td>
			<td>151807</td>
			<td>Solvent used for the synthesis of [11C]mHED</td>
		</tr>
		<tr>
			<td>Ammonium formate</td>
			<td>Sigma Aldrich</td>
			<td>798568</td>
			<td>Reagent used for the synthesis of [11C]MRB</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Ricca Chemical</td>
			<td>642-16</td>
			<td>Reagent used for the synthesis of [11C]ER-176</td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Mettler Toledo&#160;</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&#160;</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 &#8211; 80 mesh</td>
			<td>Supelco</td>
			<td>CN-10478-U</td>
			<td>Used in the FxC Pro Module</td>
		</tr>
		<tr>
			<td>Compressed &#8211; NOS (99% nitrogen/1% oxygen)</td>
			<td>Airgas&#160;</td>
			<td>CN-X02NI99C3003091</td>
			<td>Target gas used in cyclotron bombardment</td>
		</tr>
		<tr>
			<td>Dispensing hot cell (DHC)</td>
			<td>Comecer&#160;</td>
			<td>M-MIP1-1390</td>
			<td>Dispensing radiotracers</td>
		</tr>
		<tr>
			<td>Dose calibrator&#160;</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&#160;</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&#160;</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&#160; final product and&#160; 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 [11C]ER-176, [11C]MRB, [11C]mHED and [11C]PiB</td>
		</tr>
		<tr>
			<td>Helium, research grade</td>
			<td>Airgas&#160;</td>
			<td>CN-HER-300</td>
			<td>Helium tank, 99.9999% (research grade tank)</td>
		</tr>
		<tr>
			<td>Shimadzu LC-20 Series&#160;</td>
			<td>Shimadzu</td>
			<td>Various&#160;</td>
			<td>Analytical HPLC system</td>
		</tr>
		<tr>
			<td>Hydrogen, ultra high purity grade&#160;&#160;</td>
			<td>Airgas&#160;</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 [11C]CH4 to [11C]CH3I</td>
		</tr>
		<tr>
			<td>Luna 10 mm C18 (2) 100 &#197; 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 &#197; 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 [11C]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-d7</td>
			<td>Sigma Aldrich</td>
			<td>189979</td>
			<td>Solvent used for the synthesis of [11C]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&#8221;</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&#8221;, Spinal&#160;</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 CH4 oven of FxCPro Module for conversion from [11C]CO2 to [11C]CH4</td>
		</tr>
		<tr>
			<td>Siemens Eclipse 11-MeV cyclotron</td>
			<td>Siemens</td>
			<td>RDS 111</td>
			<td>Particle accelerator used to generate [11C]CO2 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 [11C]mHED and [11C]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 [11C]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 [11C]ER-176 and other components at different concentration.</td>
		</tr>
		<tr>
			<td>Sodium iodide detector</td>
			<td>Eckert &#38; 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&#160;</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&#8221;, 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&#160;</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 [11C]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

Research

JoVE Journal

Chemistry

1.0K 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

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

The presented protocols describe two enzyme-linked immunosorbent assay (ELISA) based techniques for the rapid investigation of ligand-receptor interactions: The first assay allows the determination of dissociation constant between ligand and receptor. The second assay enables a rapid screening of blocking peptides for ligand-receptor interactions.

A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and ...

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 ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...