Automated Synthesis and Uptake Analysis of D-[Methyl-11C]-Methionine

Summary

Here, we present a protocol for the synthesis of D-[methyl-¹¹C]-methionine, a metabolic positron emission tomography tracer for bacteria, and in vitro evaluation in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The synthesis involves a single synthetic step on an automated module, followed by microcentrifuge filtration.

Abstract

Positron emission tomography (PET) has emerged as a vital molecular imaging modality providing metabolic insights that are essential for the diagnosis and treatment of disease. Among the many useful applications of PET, imaging of infection has gained much traction in the last decade. To this end, we describe the synthesis and in vitro evaluation of D-[methyl-¹¹C]-methionine, a potent metabolic tracer for living bacteria. D-[methyl-¹¹C]-methionine was synthesized from [¹¹C]methyl iodide ([¹¹C]CH₃I) using an in-loop synthesis method in an automated synthesis module. The radiotracer was analyzed by chiral high-performance liquid chromatography (HPLC) to determine its radiochemical identity and purity and then subjected to in vitro analysis using a rapid method to determine uptake in bacteria cells. The workflow described demonstrates the importance of communication and time management when developing new radiotracers, especially with short half-life isotopes such as carbon-11 (¹¹C) (t_1/2 = 20.4 min).

Introduction

The use of new imaging techniques to detect infection is an emerging field and in recent years, PET imaging has been a leading innovator with several tracers specifically sensing living bacteria^{1,2,3,4,5,6,7,8}. D-[methyl-¹¹C]-methionine is a first-generation, metabolic PET tracer that images live bacterial infection by targeting peptidoglycan, a key cell wall component in both gram-positive an....

Protocol

CAUTION: In the following protocol, there are multiple manipulations that require the handling of radioactivity. It is extremely important that every interaction with radioactivity be executed in agreement with the Radiation Safety Department of the institute and the respective national guidelines. It is mandatory to minimize the exposure to ionizing radiation for the operators involved following the “as low as reasonably achievable” (ALARA) principle.

NOTE: “Current Good Man.......

Discussion

The radiosynthesis of D-[methyl-¹¹C]-methionine was performed in a commercial synthesis module using an in-loop method that yielded superior yields and purities over previously reported conventional manual approaches (>99% ee, 22% ± 13% decay-corrected radiochemical yield versus 85% ee, 20% ± 1% decay corrected radiochemical yield)⁹. When making radiotracers from either [¹¹C]carbon dioxide or [¹¹C] methyl iodide, is it imperative that a completely clo.......

Materials

Name	Company	Catalog Number	Comments
Materials and instruments for radiosynthesis of D-[11C]methionine
0.5 M NaOH	Sigma-Aldrich, St. Louis, MO, USA
C18 Sep Pak	Waters, Milford, MA, USA
D/L-Methionine	Sigma-Aldrich, St. Louis, MO, USA		Standards
D-Homocysteine	Sigma-Aldrich, St. Louis, MO, USA		Precursor
Ethanol	Sigma-Aldrich, St. Louis, MO, USA
GE Medical Systems PET Trace	GE Healthcare, Uppsala Sweden		Cyclotron
Iodine	Merck, Darmstadt, Germany
Nickel Catalyst	Shimadzu, Kyoto, Japan
Nitrogen gas + 1% Oxygen	AirGas, Radnor, PA, USA		Target Gas
Saline	Sigma-Aldrich, St. Louis, MO, USA
Sodium Hydrogen Phosphate	Sigma-Aldrich, St. Louis, MO, USA
TRACERlab FXC	GE Healthcare, Uppsala Sweden		Synthesis Module
Water	Sigma-Aldrich, St. Louis, MO, USA
Materials and instruments for quality control of D-[11C]methionine
Acetonitrile	Sigma-Aldrich, St. Louis, MO, USA
Agilent 8890	Agilent, Santa Clara, CA, USA		Analytical GC
Bioscan AR2000	Bioscan, Santa Barbara, CA, USA		RadioTLC
Chirobiotic T2 column (250 x 4.6 mm)	Astec, Chattanooga, TN, USA		Chiral HPLC Column
Water	Sigma-Aldrich, St. Louis, MO, USA
Waters 600 Controller	Waters, Milford, MA, USA		Analytical HPLC
Materials and instruments for bacterial uptake assays of D-[11C]methionine
15 mL Centrifuge Tubes	Corning, NY, USA
50 mL Bio-Reaction Tubes	Celltreat, MA, USA
E. Coli	ATCC, Manassa, VA, USA		25922
F12 Media	Thermo, Waltham, MA, USA
Genesys 20	Thermo, Waltham, MA, USA		UV/Vis Spectrometer
Hidex AMG	Hidex, Turku, Finland		Gamma Counter
Innova 42	Brunswick, Lake Forest, IL, USA		Incubator/Shaker
LB Agar Plates	Teknova, Hollister, CA, USA
LB Broth	Teknova, Hollister, CA, USA
P. Aeruginosa	ATCC, Manassa, VA, USA		10145
S. Aureus	ATCC, Manassa, VA, USA		12600
Spin-X Filter Tubes	Corning, NY, USA
UV Cuvettes	Fisher, Waltham, MA, USA