Name: radiosynthesis of 1-(2-[18f]fluoroethyl)-l-tryptophan using a one-pot, two-step protocol
Uploaded: 2021-09-21T14:45:00
Description: Watch this Scientific Journal Video about Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol at JoVE.com

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 ...

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 ...

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