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

Summary

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.

Abstract

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, α-[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 ± 5% (decay corrected at the end of synthesis, n > 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.

Introduction

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 effects^1,2. Abnormal tryptophan metabolism is impli....

Protocol

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 'as low as reasonably achievable' (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 contaminate....

Results

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 °C for 10 min.

Before receiving [

Discussion

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 synthesis^38,39, detecting and grading tumors⁴⁰, guiding epilepsy surgery^41,42.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[18F]Fluoride in [18O]H2O	PETNET Solutions Inc.	N/A
4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane	ACROS	291950010	Kryptofix 222 or K222, 98%
Acetic acid	ACROS	222142500	99.8%
Acetonitrile	Sigma-Aldrich	271004	anhydrous, 99.8%
Agilent 1260 HPLC system	Agilent Technologies	Agilent 1260	Agilent 1260 series
Analytcial chiral HPLC column	Sigma-Aldrich	12024AST	Astec CHIROBIOTIC T, 25 cm × 4.6 mm
Carbon dioxide, 60 LBS	Airgas	REFR744R200S	99.99%
D-FETrp standard reference	Affinity Research Chemicals Inc	N/A	Custom synthesis
Empty sterile vial	Jubilant HollisterStier	7515	20 mm closure, 10 mL
Ethanol	Decon Labs	2716	200 proof, USP grade. ≥99.9%
Fisherbrand 13 mm Syringe Filter, 0.22 µm, PVDF, sterile	Fisher Scientific	09-720-3
Hydrochloric acid	Sigma-Aldrich	30721	≥37%
Isopropanol	Decon Labs	8316	70%, sterile
L-[18F]FETrp radiolabeling precursor	Affinity Research Chemicals Inc	N/A	Custom synthesis
L-FETrp standard reference	Affinity Research Chemicals Inc	N/A	Custom synthesis
Light C8 cartridge	Waters	WAT036770	Sep-Pak  C8 plus light cartridge
Needle, 20 G x 1	Becton-Dickinson & Co.	305175
Needle, 20 G x 1 ½	Becton-Dickinson & Co.	305176
Needle, 21 G x 2	Becton-Dickinson & Co.	305129
Neutral aluminum oxide	Waters	WAT023561	Sep-Pak alumina N plus light
Nylon membrane (0.20 µm )	MilliPore	GNWP04700	47 mm
Pall Acrodisc 25 mm syringe sterile filter	Pall Corporation	4907
PETCHEM radiochemistry synthesis system	PETCHEM Solutions Inc. Pinckney, MI	N/A	Radiosynthesizer
pH strips 2.0 - 9.0	EMD Millipore	1.09584.0001
Potassium carbonate	Sigma-Aldrich	367877	99.995%
Quaternary methylammonium light cartridge	Waters	186004051	Sep-Pak QMA light
Semi-preparative C18 HPLC column	Phenomenex	00D-4253-N0	100 × 10 mm
Semi-preparative chiral HPLC column	Sigma-Aldrich	12034AST	Astec CHIROBIOTIC T, 25 cm × 10 mm
Sodium chloride injection 23.4%	APP Pharmaceutical, LLC	18730	USP grade
Sodium chloridei injection 0.9%	Hospira	NDC 0409-4888-10	USP grade
Sodium hydroxide	Honeywell	306576	99.99%
Spinal needle, 20 G x 3 ½	Becton-Dickinson & Co.	405182
Sterile alcohol prep pads	BioMed Resource Inc.	PC661
Sterile empty vials, 2 mL	Hollister Stier	7505ZA	13 mm closure
Sterile empty vials, 30 mL	Jubilant HollisterStier	7520ZA	20 mm closure
Syringe PP/PE, 3 mL, Luer Lock	Air-Tite	4020-X00V0
Syringe PP/PE, 5 mL, Luer Lock	Becton-Dickinson & Co.	309646
Syringe,  PP/PE, 10 mL, NORM-JECT	Air-Tite	4100-000V0
Syringe, 1 mL, Luer Slip	Becton-Dickinson & Co.	309659
Syringe, 3 mL, Luer-Lock	Becton-Dickinson & Co.	309657
Ultra high purity argon	Airgas	AR UHP300	99.999%
Ultrapure water	MilliporeSigma	ZRQSVP300	Direct-Q 3 tap to pure and ultrapure water purification system