This work was supported by the Diagnostic &#38; Research PET/MRI Center, and by the Departments of Biomedical Research and Radiology at Nemours/Alfred I. duPont Hospital for Children.

CAUTION: The protocol involves radioactive materials. Any additional dose of radioactive materials could lead to a proportional increase in the chance of adverse health effects such as cancer. Researchers must follow the &#39;as low as reasonably achievable&#39; (ALARA) dose practices to guide the radiosynthesis protocol with adequate protection in the hot cell or lead hood. Minimizing direct contact time, using a lead shield, and keeping maximum distance for any radiation exposure step in the radiosynthesis process are essential. Wear a radiation dosimetry badge and hand monitoring rings throughout the entire experiment, and frequently monitor potentially contaminated surfaces such as gloves, sleeves, and feet. Nuclear Regulatory Commission (NRC), local, and institutional regulations must be followed for the usage, shipping, and disposal of any radioactive materials.
1. Initial preparations
<ol>
	<li>Prepare 10% ethanol in 50 mM sodium acetate/acetic acid mobile phase for semipreparative high-performance liquid chromatography (HPLC).
	<ol>
		<li>Place 3 mL of glacial acetic acid in a clean 1000 mL volumetric flask. Add 900 mL of ultrapure water (18 million ohm-cm resistivity at 25 &#176;C) into the volumetric flask; add approximately 8 mL of 6 M sodium hydroxide solution and adjust the pH to 5.5 using a calibrated pH meter and a pH strip. After the solution cools down to room temperature, make up the volume to 1000 mL with ultrapure water to prepare the 50 mM sodium acetate/acetic acid (pH 5.5) solution.</li>
		<li>Vacuum-filter the solution through a 0.2 &#181;m membrane filter and transfer the solution to two 500 mL solvent bottles.</li>
		<li>Place approximately 250 mL of the above buffer in a 500 mL volumetric flask. Use a graduated cylinder to measure 50 mL of United States Pharmacopeia (USP) ethanol and add the ethanol to the volumetric flask. Make up the volume to 500 mL with 50 mM sodium acetate/acetic acid, and measure the pH value with a pH strip.</li>
	</ol>
	</li>
	<li>Prepare quality control (QC) solutions for a system suitability test.
	<ol>
		<li>Refill the HPLC solvent bottles with fresh ultrapure water (solvent A) and ethanol (solvent B). Prime the HPLC pump and load the HPLC program with a flow rate of 1 mL/min consisting of 30% solvent A and 70% solvent B (v/v).</li>
		<li>Make a control solution (blank solution) for QC. Add 5 mL of 0.9% sodium chloride into a 20 mL glass vial. Add 0.15 mL of 23.4% sodium chloride into the above solution. Add 6 mL of semipreparative HPLC mobile phase prepared in step 1.1.3 (10% ethanol in 50 mM sodium acetate/acetic acid, pH 5.5) to the glass vial.</li>
		<li>Prepare 1 &#181;g/mL of nonradiolabeled L-FETrp and 5 &#181;g/mL of racemic L-FETrp and D-FETrp mixtures (standard solutions).</li>
		<li>Build a calibration curve using standard L-FETrp solutions (0.1 &#181;g/mL, 0.5 &#181;g/mL, 1.0 &#181;g/mL, 10 &#181;g/mL, 100 &#181;g/mL).</li>
		<li>Set up the HPLC sequence. Ensure that the sequence includes one run of the blank sample solution, two runs of the standard L-FETrp (1 &#181;g/mL), one run of the racemic L-FETrp and D-FETrp mixtures (5 &#181;g/mL), and one run of the final radiopharmaceutical.</li>
		<li>Run the partial HPLC sequence to test the system suitability before analyzing the radioactive samples using an analytical HPLC column (250 x 4.6 mm).
		<ol>
			<li>Run one blank sample and ensure that the chromatogram of the blank sample shows no or insignificant peaks between the void volume and 10 min of the chromatogram.</li>
			<li>Run two replicates of the standard solution (contains 1 &#181;g/mL of L-FETrp). Ensure that the areas of the L-FETrp in the two replicates are within &#177;5% of the mean value.</li>
			<li>Run one sample of the L-FETrp and D-FETrp mixtures (5 &#181;g/mL of L-FETrp and D-EFTrp, respectively). Ensure that L-FETrp and D-FETrp can be identified on the chromatogram and baseline resolved.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare the radiolabeling solutions and other supplies.
	<ol>
		<li>Add the following solutions to five 1.5-mL V-shaped vials, respectively. Vial 1: 1 mL of potassium carbonate (K2CO3)/K222 solution (5 mg/mL K222 and 1 mg/mL K2CO3 in a water/acetonitrile solution, 1/99, v/v) for [18F]fluoride elution; Vial 2: 0.4 mL of anhydrous acetonitrile for [18F]fluoride drying; Vial 3: radiolabeling precursor in anhydrous acetonitrile (1-2 mg in 0.5 mL of anhydrous acetonitrile) for [18F]fluoride incorporation; Vial 4: hydrochloric acid (2 M, 0.25 mL) in acetonitrile (0.25 mL) for acidolysis; Vial 5: 2 M sodium hydroxide (0.25 mL) for neutralization of the reaction mixtures.</li>
		<li>Activate a quaternary methylammonium (QMA) light cartridge by first passing through 10 mL of saturated sodium bicarbonate solution, followed by 10 mL of ultrapure water, and then flush the cartridge with a nitrogen flow. Condition a light C8 cartridge and a neutral aluminum oxide cartridge by passing through 10 mL of ethanol, followed by 10 mL of ultrapure water.</li>
		<li>Add 0.15 mL of 23.4% sodium chloride and 5 mL of 0.9% sodium chloride to a 30 mL sterile formulation vial to adjust the tonicity and dilute the HPLC fraction.</li>
		<li>Prepare a solution (1 mL of sodium acetate/acetic acid buffer, 50 mM, pH = 5.5 prepared in step 1.1.1, 1 mL of ethanol, and 0.5 mL of water [total 2.5 mL]) in a syringe for rinsing the reaction vessel; load into a 10 mL sterile vial.</li>
	</ol>
	</li>
</ol>
2. Assemble the radiolabeling supplies and radiosynthesize L-[18F]FETrp
<ol>
	<li>Assemble the radiolabeling supplies.
	<ol>
		<li>Turn on the module power, carbon dioxide, compressed air, argon lines, and the programmable logic controller (PLC) power. Click the mod_pscf18 button to activate the program of the radiochemistry synthesis system. Initialize the input, output, and formulation MVP, and ensure that the MVPs are at positions 4, 1, 1, respectively.</li>
		<li>Ensure that the HPLC loop is in the Inject position and the QMA light cartridge in the [18F]fluoride trapping position.</li>
		<li>Install the semipreparative HPLC mobile phase bottle (containing the solution prepared in step 1.1.3). Equilibrate the HPLC system by passing the mobile phase through the chiral HPLC column (250 x 10 mm) and C18 column (100 x 10 mm) at a flow rate of 2 mL/min for at least 30 min, then switch the diversion valve, let the HPLC mobile pass through the chiral HPLC column only, and increase the flow rate to 3 mL/min.</li>
		<li>Install the QMA light cartridge in the [18F]fluoride trapping/releasing line. Install the stacked alumina/C8 cartridges between the input MVP position 6 and the intermediate vial, which is a 10 mL V-shaped vial connected to the HPLC sample loop. Install a 10 mL empty vial (venting vial) to the output MVP position 4 with another needle attached to the vial as a vent. Install the reagent vials 1-5 to the input MVP positions 1-5, respectively.</li>
		<li>Install the vial containing the rinse solution prepared in step 1.3.4 to the output MVP position 6.</li>
		<li>Install a 500 mL waste bottle (to collect the HPLC waste passing through both the chiral and C18 columns) to the formulation MVP position 1. Install another 500 mL waste bottle (to collect HPLC waste passing through the chiral column) to the waste collection end of the four-port two-position valve.</li>
		<li>Connect the fraction collection vial (prefilled solution prepared in step 1.3.3) to the formulation MVP position 2. Connect the output MVP position 3 (gas line) and final product delivery line to the formulation MVP position 2 vials to recover the final formulated product. Install a 10 mL empty sterile vial in formulation MVP position 3, which will be used as the backup vial for the target fraction collection.</li>
		<li>Install the C18 short column between the four-port, two-position diversion valve and the formulation MVP. 
		NOTE: During the installation of the reagent and formulation vials, ensure the argon supply in the control panel is off, and the argon pressure is zero to avoid any unexpected liquid transfer during the vial assembly. Double-check all needle connections, vial positions, and MVP positions for reproducible radiosynthesis.</li>
	</ol>
	</li>
	<li>Radiosynthesis of L-[18F]FETrp
	<ol>
		<li>Receive and survey [18F]fluoride.
		<ol>
			<li>When receiving the [18F]fluoride solution (15 &#177; 3 gigabecquerel (GBq) at the start of synthesis, see the Table of Materials), survey the lead box on the surface and at 1 m to record the maximum radiation exposure rates. Do a wipe test to ensure the shipping box is not contaminated. Record the [18F]fluoride dose and time.</li>
		</ol>
		</li>
		<li>Transfer [18F]fluoride.
		<ol>
			<li>Transfer the radioactivity to the radiochemistry synthesis system.</li>
			<li>Connect the argon line with a short needle and the [18F]fluoride transfer line with a long needle to the [18F]fluoride vial. Close the hot-cell glass door and lead door. 
			CAUTION: Use a long clamp to push down both needles; ensure the long needle tip sits in the bottom of the [18F]fluoride vial so that all [18F]fluoride can be transferred out. Typically, a V-shaped vial is requested for the [18F]fluoride delivery.</li>
		</ol>
		</li>
		<li>Trap, elute, and azeotropically dry [18F]fluoride.
		<ol>
			<li>Click Ar Supply to turn on the argon supply line, increase argon pressure, turn on the [18F]fluoride pushing line, and push the aqueous [18F]fluoride through the QMA light cartridge. After all the radioactivity is trapped in the QMA light cartridge, and the radioactivity detector reading is steady, increase the argon pressure and blow argon through the cartridge for another 5 min to remove excess water.</li>
			<li>Turn off the [18F]fluoride pushing line, decrease the argon pressure to zero, switch the six-port two-position valve from the [18F]fluoride trapping position to the elution position. Open the reaction vial, turn on the input MVP position 1 argon line, push the K222/K2CO3 solution into the input MVP position 1 vial through the QMA light cartridge to elute out the radioactivity into the reaction vial. Switch the [18F]fluoride elution position to the trapping position.</li>
			<li>Click the Heat button to heat the reactor at 110 &#176;C, turn on the output MVP position 4 argon line (sweeping line) that connects to the reactor, and evaporate the solvent into the output MVP position 4 vial.</li>
			<li>Click the Cool button to cool down the reactor to room temperature with compressed carbon dioxide, turn off the sweeping line, switch the input MVP position 1 to position 2, and add the anhydrous acetonitrile in the vial 2. Turn on the sweeping line and heater to azeotropically dry [18F]fluoride at 110 &#176;C.</li>
		</ol>
		</li>
		<li>Add the radiolabeling precursor and incorporate [18F]fluoride.
		<ol>
			<li>Cool down the reactor to room temperature, turn off the sweeping line, switch the input MVP position 2 to position 3, and add the tosylate radiolabeling precursor in vial 3. Close the reactor, and heat the reaction mixtures at 100 &#176;C for 10 min.</li>
		</ol>
		</li>
		<li>Evaporate the reaction solvent and acidolyse.
		<ol>
			<li>Cool down and open the reactor, turn on the sweeping line, and evaporate the reaction solvent at 100 &#176;C.</li>
			<li>Cool down the reactor, turn off the sweeping line, switch the input MVP position 3 to position 4, and add the hydrochloric acid/acetonitrile mixture (0.5 mL, 1/1, v/v). Heat the reaction at 100 &#176;C for 10 min to deprotect the tert-butyl and tert-butyloxycarbonyl-protecting groups in the radiolabeling precursor.</li>
		</ol>
		</li>
		<li>Neutralize the reaction.
		<ol>
			<li>Cool down the reactor to room temperature, and switch the input MVP position 4 to position 5 to add 2 M sodium hydroxide to neutralize the reaction mixture. Turn off the input MVP argon line.</li>
		</ol>
		</li>
		<li>Transfer the reaction mixture to the intermediate vial.
		<ol>
			<li>Click the F button in the output MVP to switch the output MVP from the venting position 4 to position 5, and then click the F button in the input MVP to switch the input MVP from position 5 to position 6. Turn on the output MVP argon line. Push the reaction mixture through the stacked neutral aluminum oxide and C8 cartridges to the intermediate vial installed before the HPLC sample loop.</li>
		</ol>
		</li>
		<li>Rinse the reactor and transfer the solution.
		<ol>
			<li>Switch the output MVP position 5 to position 6, push the rinse solution in the vial of output MVP position 6 through the reaction vial, and cartridges to the intermediate vial, successively. Note that the volume of the combined mixture is approximately 3.5 mL. Close the reaction vessel.</li>
		</ol>
		</li>
		<li>Load the combined mixture to the HPLC loop and purify the mixture.
		<ol>
			<li>Switch the HPLC loop from injection position to load position, turn on the input MVP argon line, and load the mixtures to the 5 mL HPLC loop. When the load is complete, switch the load button to inject, and click Inject HPLC to start the HPLC chromatogram at a flow rate of 3 mL/min. Click the HPLC monitor button to access the real-time HPLC chromatogram.</li>
		</ol>
		</li>
		<li>Divert the target fraction to the C18 column.
		<ol>
			<li>Click the diversion MVP button to divert the target HPLC fraction to the short C18 column at approximately 12 min.</li>
		</ol>
		</li>
		<li>Flush the chiral HPLC column and purify the fraction in the C18 column.
		<ol>
			<li>After the target radioactivity is collected in the C18 column (and the HPLC mobile phase flows into the formulation MVP position 1 waste bottle), switch the four-port two-position diversion valve to the waste collection position. Flush the chiral column at 4 mL/min for 6 min to remove the minor D-[18F]FETrp enantiomer and other ultraviolet (UV) impurities.</li>
			<li>Click the diversion MVP button to divert the HPLC mobile phase back to the formulation MVP position 1 at a flow rate of 3 mL/min. Observe that the mobile phase passes through the chiral column and C18 column to purify L-[18F]FETrp retained in the C18 column.</li>
		</ol>
		</li>
		<li>Collect the target fraction.
		<ol>
			<li>Collect the eluent at approximately 32-34 min into the formulation MVP position 2 vial. 
			NOTE: Typically, a 2 min HPLC fraction is collected, and the total volume plus the prefilled sodium chloride solution is 8-15 mL.</li>
		</ol>
		</li>
		<li>Deliver the dose to a sterile final product vial.
		<ol>
			<li>Push the solution in the formulation MVP position 2 vial through the delivery line and sterile filter into the final dose vial (via the preinstalled sterile venting filter needle). 
			NOTE: Protocol steps 2.2.9-2.2.13 are steps used for the HPLC purification of L-[18F]FETrp.</li>
		</ol>
		</li>
		<li>Assay the radioactivity and dose volume.
		<ol>
			<li>Remove the sterile filter and assay the final dose activity and volume.</li>
			<li>Flush the system with ultrapure water followed by ethanol at 4 mL/min for at least 15 min each. Turn off the HPLC pump and PLC box; close the program; shut down the compressed air line, argon line, and carbon dioxide line; and shut down the main power of the module.</li>
		</ol>
		</li>
		<li>Withdraw the QC dose and run the QC samples.
		<ol>
			<li>Withdraw approximately 0.1 mL of the final dose into a 0.2 mL insert of a QC vial. Run the hot sample as the &#34;partial sequence&#34; program following the system suitability test described in step 1.2.6 .</li>
		</ol>
		</li>
		<li>Analyze the QC data and release the dose.
		<ol>
			<li>Calculate the chemical and radiochemical purities, enantiomeric excess value, and molar activity; determine the pH value.</li>
			<li>Release the dose if all testing results pass the acceptable range.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Post-run system clean
<ol>
	<li>Survey the radiolabeling module using a Geiger-Mueller (GM) survey meter to ensure that the radioactivity is adequately decayed (at least 24 h) before the system clean.</li>
	<li>Turn on the radiolabeling module power and PLC box power; activate the program of the radiochemistry synthesis system; and initialize the input MVP, output MVP, and formulation MVP. Switch the column selector to the bypass position.</li>
	<li>Detach the QMA light, alumina, and C8 cartridges.</li>
	<li>Flush all vials and lines with ultrapure water first, followed by ethanol. Dry the vials and lines with high-purity argon.</li>
	<li>Seal each line vent using a sterile needle with a cap. Replace the reagent vials and reaction vessel with oven-burned vials and reaction vessel, respectively.</li>
	<li>Turn off the HPLC pump and PLC box; close the program; shut down the compressed air line, argon line, and carbon dioxide line; and shut down the main power of the module.</li>
</ol>

The authors declare that no competing financial interests exist.

Tryptophan is an essential amino acid for humans. It plays an important role in the regulation of mood, cognitive function, and behavior. Radiolabeled tryptophan derivatives, particularly the carbon-11-labeled [11C]AMT, have been extensively studied due to their unique role in mapping serotonin synthesis38,39, detecting and grading tumors40, guiding epilepsy surgery41,42...

In humans, tryptophan is an essential component of the daily diet. Tryptophan is primarily metabolized via the kynurenine pathway (KP). The KP is catalyzed by two rate-limiting enzymes, indoleamine 2, 3-dioxygenase (IDO) and tryptophan 2, 3-dioxygenase (TDO). More than 95% of tryptophan is converted into kynurenine and its downstream metabolites, ultimately generating nicotinamide adenine dinucleotide, which is essential to cellular energy transduction. The KP is a key regulator of the immune system and an important regulator of neuroplasticity and neurotoxic effects1,2. Abnormal tryptophan metabolism is implicated in various neurologic, oncologic, psychiatric, and metabolic disorders; therefore, radiolabeled tryptophan analogs have been extensively used in clinical investigation. The two most common clinically investigated tryptophan radiotracers are 11C-&#945;-methyl-L-tryptophan ([11C]AMT) and 11C-5-hydroxytryptophan (11C-5-HTP)3.
In the 1990s, 11C-5-HTP was used to visualize serotonin-secreting neuroendocrine tumors4 and to diagnose and monitor therapy of metastatic hormone-refractory prostatic adenocarcinoma5. Later, it was used as an imaging tool for the quantification of the serotonergic system in the endocrine pancreas6. 11C-5-HTP has also been a promising tracer for noninvasive detection of viable islets in intraportal islet transplantation and type 2 diabetes7,8. Over the past two decades, many radiolabeled amino acids have advanced to clinical investigation9,10. In particular, the carbon-11-labeled tryptophan analog [11C]AMT has received extensive attention for mapping brain serotonin synthesis11,12,13,14 and for localizing epileptic foci, epileptogenic tumors, tuberous sclerosis complex, gliomas, and breast cancers15,16,17,18,19,20,21,22,23,24,25,26. [11C]AMT also has high uptake in various low- and high-grade tumors in children27. Furthermore, kinetic tracer analysis of [11C]AMT in human subjects has been used to differentiate and grade various tumors and differentiate glioma from radiation-induced tissue injury15. [11C]AMT-guided imaging shows significant clinical benefits in brain disorders3,25. However, due to the short half-life of carbon-11 (20 min) and the laborious radiosynthesis procedures, [11C]AMT use is restricted to the few PET centers with an onsite cyclotron and a radiochemistry facility.
Fluorine-18 has a favorable half-life of 109.8 min, compared with the 20 min half-life of carbon-11. Increasingly, efforts have been focused on the development of fluorine-18-labeled radiotracers for tryptophan metabolism3,28. A total of 15 unique fluorine-18 radiolabeled tryptophan radiotracers have been reported in terms of radiolabeling, transport mechanisms, in vitro and in vivo stability, biodistribution, and tumor uptake in xenografts. However, rapid in vivo defluorination was observed for several tracers, including 4-, 5-, and 6-[18F]fluorotryptophan, precluding further clinical translation29. 5-[18F]Fluoro-&#945;-methyltryptophan (5-[18F]FAMT) and 1-(2-[18F]fluoroethyl)-L-tryptophan (L-[18F]FETrp, also known as (S)-2-amino-3-(1-(2-[18F]fluoroethyl)-1H-indol-3-yl)propanoic acid, molecular weight 249.28 g/mole), are the two most promising radiotracers with favorable in vivo kinetics in animal models and great potential to surpass [11C]AMT for the evaluation of clinical conditions with deregulated tryptophan metabolism28. 5-[18F]FAMT showed high uptake in IDO1-positive tumor xenografts of immunocompromised mice and is more specific to imaging the KP than [11C]AMT28,30. However, the in vivo stability of 5-[18F]FAMT remains a potential concern as no in vivo defluorination data have been reported beyond 30 min post injection of the tracer30.
A preclinical study in a genetically engineered medulloblastoma mouse model showed that when compared with 18F-fluorodeoxyglucose (18F-FDG), L-[18F]FETrp had high accumulation in brain tumors, negligible in vivo defluorination, and low background uptake, demonstrating a superior target-to-nontarget ratio31,32. Radiation dosimetry studies in mice indicated that L-[18F]FETrp had an approximately 20% lower favorable dosimetry exposure than the clinical 18F-FDG PET tracer33. In agreement with other researchers&#39; findings, preclinical study data provide substantial evidence to support the clinical translation of L-[18F]FETrp for the investigation of abnormal tryptophan metabolism in humans with brain disorders such as epilepsy, neuro-oncology, autism, and tuberous sclerosis28,31,32,33,34,35,36. An overall comparison between the three most widely investigated tracers for tryptophan metabolism, 11C-5-HTP, [11C]AMT, and L-[18F]FETrp, is shown in Table 1. Both 11C-5-HTP and [11C]AMT have a short half-life and laborious radiolabeling procedures. A protocol for the radiosynthesis of L-[18F]FETrp using a one-pot, two-step approach is described here. The protocol features the use of a small amount of radiolabeling precursor, a small volume of reaction solvents, low loading of toxic K222, and an environmentally benign and injectable mobile phase for purification and easy formulation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[18F]Fluoride in [18O]H2O</td>
			<td>PETNET Solutions Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane</td>
			<td>ACROS</td>
			<td>291950010</td>
			<td>Kryptofix 222 or K222, 98%</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>ACROS</td>
			<td>222142500</td>
			<td>99.8%</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004</td>
			<td>anhydrous, 99.8%</td>
		</tr>
		<tr>
			<td>Agilent 1260 HPLC system</td>
			<td>Agilent Technologies</td>
			<td>Agilent 1260</td>
			<td>Agilent 1260 series</td>
		</tr>
		<tr>
			<td>Analytcial chiral HPLC column</td>
			<td>Sigma-Aldrich</td>
			<td>12024AST</td>
			<td>Astec CHIROBIOTIC T, 25 cm &#215; 4.6 mm</td>
		</tr>
		<tr>
			<td>Carbon dioxide, 60 LBS</td>
			<td>Airgas</td>
			<td>REFR744R200S</td>
			<td>99.99%</td>
		</tr>
		<tr>
			<td>D-FETrp standard reference</td>
			<td>Affinity Research Chemicals Inc</td>
			<td>N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Empty sterile vial</td>
			<td>Jubilant HollisterStier</td>
			<td>7515</td>
			<td>20 mm closure, 10 mL</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td>200 proof, USP grade. &#8805;99.9%</td>
		</tr>
		<tr>
			<td>Fisherbrand 13 mm Syringe Filter, 0.22 &#181;m, PVDF, sterile</td>
			<td>Fisher Scientific</td>
			<td>09-720-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>30721</td>
			<td>&#8805;37%</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Decon Labs</td>
			<td>8316</td>
			<td>70%, sterile</td>
		</tr>
		<tr>
			<td>L-[18F]FETrp radiolabeling precursor</td>
			<td>Affinity Research Chemicals Inc</td>
			<td>N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>L-FETrp standard reference</td>
			<td>Affinity Research Chemicals Inc</td>
			<td>N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Light C8 cartridge</td>
			<td>Waters</td>
			<td>WAT036770</td>
			<td>Sep-Pak&#160; C8 plus light cartridge</td>
		</tr>
		<tr>
			<td>Needle, 20 G x 1</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 20 G x 1 &#189;</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>305176</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 21 G x 2</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>305129</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral aluminum oxide</td>
			<td>Waters</td>
			<td>WAT023561</td>
			<td>Sep-Pak alumina N plus light</td>
		</tr>
		<tr>
			<td>Nylon membrane (0.20 &#181;m )</td>
			<td>MilliPore</td>
			<td>GNWP04700</td>
			<td>47 mm</td>
		</tr>
		<tr>
			<td>Pall Acrodisc 25 mm syringe sterile filter</td>
			<td>Pall Corporation</td>
			<td>4907</td>
			<td></td>
		</tr>
		<tr>
			<td>PETCHEM radiochemistry synthesis system</td>
			<td>PETCHEM Solutions Inc. Pinckney, MI</td>
			<td>N/A</td>
			<td>Radiosynthesizer</td>
		</tr>
		<tr>
			<td>pH strips 2.0 - 9.0</td>
			<td>EMD Millipore</td>
			<td>1.09584.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>367877</td>
			<td>99.995%</td>
		</tr>
		<tr>
			<td>Quaternary methylammonium light cartridge</td>
			<td>Waters</td>
			<td>186004051</td>
			<td>Sep-Pak QMA light</td>
		</tr>
		<tr>
			<td>Semi-preparative C18 HPLC column</td>
			<td>Phenomenex</td>
			<td>00D-4253-N0</td>
			<td>100 &#215; 10 mm</td>
		</tr>
		<tr>
			<td>Semi-preparative chiral HPLC column</td>
			<td>Sigma-Aldrich</td>
			<td>12034AST</td>
			<td>Astec CHIROBIOTIC T, 25 cm &#215; 10 mm</td>
		</tr>
		<tr>
			<td>Sodium chloride injection 23.4%</td>
			<td>APP Pharmaceutical, LLC</td>
			<td>18730</td>
			<td>USP grade</td>
		</tr>
		<tr>
			<td>Sodium chloridei injection 0.9%</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-10</td>
			<td>USP grade</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Honeywell</td>
			<td>306576</td>
			<td>99.99%</td>
		</tr>
		<tr>
			<td>Spinal needle, 20 G x 3 &#189;</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>405182</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pads</td>
			<td>BioMed Resource Inc.</td>
			<td>PC661</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile empty vials, 2 mL</td>
			<td>Hollister Stier</td>
			<td>7505ZA</td>
			<td>13 mm closure</td>
		</tr>
		<tr>
			<td>Sterile empty vials, 30 mL</td>
			<td>Jubilant HollisterStier</td>
			<td>7520ZA</td>
			<td>20 mm closure</td>
		</tr>
		<tr>
			<td>Syringe PP/PE, 3 mL, Luer Lock</td>
			<td>Air-Tite</td>
			<td>4020-X00V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe PP/PE, 5 mL, Luer Lock</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe,&#160; PP/PE, 10 mL, NORM-JECT</td>
			<td>Air-Tite</td>
			<td>4100-000V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1 mL, Luer Slip</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 3 mL, Luer-Lock</td>
			<td>Becton-Dickinson &#38; Co.</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra high purity argon</td>
			<td>Airgas</td>
			<td>AR UHP300</td>
			<td>99.999%</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>MilliporeSigma</td>
			<td>ZRQSVP300</td>
			<td>Direct-Q 3 tap to pure and ultrapure water purification system</td>
		</tr>
	</tbody>
</table>

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.

The reaction scheme is shown in Figure 1. The radiolabeling includes the following two steps: 1) reaction of the tosylate radiolabeling precursor with [18F]fluoride provides the 18F-labeled intermediate, and 2) deprotection of the tert-butyloxycarbonyl and tert-butyl-protecting groups in the intermediate affords the final product L-[18F]FETrp. Both reaction steps continue at 100 &#176;C for 10 min.
Before receivin...

Watch this Scientific Journal Video about Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol at JoVE.com

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.

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 ...

radiosynthesis-1-2-18ffluoroethyl-l-tryptophan-using-one-pot-two-step

Research

JoVE Journal

Chemistry

3.0K Views.  Nemours/Alfred I. duPont Hospital for Children. 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.

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

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

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 ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

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 ...

A Protocol for Electrochemical Evaluations and State of Charge Diagnostics of a Symmetric Organic Redox Flow Battery

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid and deployment of renewable energy technologies. Among the many flow battery chemistries, non-aqueous flow batteries have the potential to achieve high energy density because of the broad voltage windows of non-aqueous electrolytes. However, significant technical hurdles exist currently limiting non-aqueous flow batteries to demonstrate their full potential, such as low redox concentrations, low operating currents, under-explored battery status monitoring, etc. In an attempt to address these limitations, we recently reported a non-aqueous flow battery based on a highly soluble, redox-active organic nitronyl nitroxide radical compound, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). This redox material exhibits an ambipolar electrochemical property, and therefore can serve as both anolyte and catholyte redox materials to form a symmetric flow battery chemistry. Moreover, we demonstrated that Fourier transform infrared (FTIR) spectroscopy could measure the PTIO concentrations during the PTIO flow battery cycling and offer reasonably accurate detection of the battery state of charge (SOC), as cross-validated by electron spin resonance (ESR) measurements. Herein we present a video protocol for the electrochemical evaluation and SOC diagnosis of the PTIO symmetric flow battery. With a detailed description, we experimentally demonstrated the route to achieve such purposes. This protocol aims to spark more interests and insights on the safety and reliability in the field of non-aqueous redox flow batteries.

We present the protocols for electrochemically evaluating a symmetric non-aqueous organic redox flow battery and for diagnosing its state of charge using FTIR.

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Separation of Aldehydes and Reactive Ketones from Mixtures Using a Bisulfite Extraction Protocol

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer by generating a charged structure provides an opportunity to use extraction as a simple purification technique. By combining the use of a miscible organic solvent with saturated sodium bisulfite, aldehydes and reactive ketones can be successfully transformed into charged bisulfite adducts that can then be separated from other organic components of a mixture by the introduction of an immiscible organic layer. Here, we describe a simple protocol for the removal of aldehydes, including sterically-hindered neopentyl aldehydes and some ketones, from chemical mixtures. Ketones can be separated if they are sterically unhindered cyclic or methyl ketones. For aliphatic aldehydes and ketones, dimethylformamide is used as the miscible solvent to improve removal rates. The bisulfite addition reaction can be reversed by basification of the aqueous layer, allowing for the re-isolation of the reactive carbonyl component of a mixture.

Here, we present a protocol to remove aldehydes and reactive ketones from mixtures by a liquid-liquid extraction protocol directly with saturated sodium bisulfite in a miscible solvent. This combined protocol is rapid and facile to perform. The aldehyde or ketone can be re-isolated by the basification of the aqueous layer.

The purification of organic compounds is an essential component of routine synthetic operations. The ability to remove contaminants into an aqueous layer ...

A Two-Step Protocol for Umpolung Functionalization of Ketones Via Enolonium Species

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. Recently, we have developed a two-step strategy for ketone enolate umpolung that has enabled the development of methods for chlorination, azidation, and amination using azoles. In addition, we have developed C-C bond&#8211;forming arylation and allylation reactions. At the heart of these methods is the preparation of the intermediate and highly reactive enolonium species prior to addition of a reactive nucleophile. This strategy is thus reminiscent of the preparation and use of metal enolates in classical synthetic chemistry. This strategy allows the use of nucleophiles that would otherwise be incompatible with the strongly oxidizing hypervalent iodine reagents. In this paper we present a detailed protocol for chlorination, azidation, N-heteroarylation, arylation, and allylation. The products include motifs prevalent in medicinally active products. This article will greatly assist others in using these methods.

A two step one-pot protocol for the umpolung of ketone enolates to enolonium species and addition of a nucleophile to the &#945;-position is described. Nucleophiles include chloride, azide, azoles, allyl-silanes, and aromatic compounds.

&#945;-Functionalization of ketones via umpolung of enolates by hypervalent iodine reagents is an important concept in synthetic organic chemistry. ...

Methods Article

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