Sensing Living Bacteria in Vivo Using d-Alanine-Derived 11C Radiotracers

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-11C]Alanine and the dipeptide d-[3-11C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [11C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-11C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[18F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-11C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-11C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-11C]alanine to address a number of important human infections.


TABLE OF CONTENTS
All chemical reagents were purchased from commercial sources (Acros Organics, Alfa Aesar, & Sigma-Aldrich) and used without further purification unless otherwise stated. All separatory cartridges were purchased from Waters. All reactions were performed under inert atmosphere of dry nitrogen and monitored by thin layer chromatography (TLC) on precoated (250 µm) silica gel 60 F254 aluminum sheets and visualized under a UV-254 lamp followed by staining with cerium ammonium molybdate or potassium permanganate. Flash chromatography was performed on silica gel (60Å pore size). 1 H and 13 C NMR spectra were obtained on a Bruker Avance III HD 400 mHz instrument at the UCSF Nuclear Magnetic Resonance Laboratory and data were processed using the accompanying Topspin software package. Chemical shifts (d) were reported in ppm relative to known solvent (7.28 ppm for CDCl3, 3.33 ppm for MeOD, & 2.5 ppm for DMSO-d6). Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). High resolution mass spectra (HRMS) services were provided by Notre Dame Mass Spectrometry & Proteomics Facility. Analytical HPLC was performed using a Waters pump equipped with a manual Rheodyne injector (1 mL loop) and a UV detector and a RAD detector. Reversed-phase chromatography used a Phenomenex Luna C18 column stationary phase, a mobile phase of 70:30 acetonitrile/4 mM sodium formate (aqueous) at a flowrate of 1 mL/min, and a 254 nm observation wavelength. Chiral chromatography used a Phenomenex Chirex 3126 column stationary phase, a mobile phase of 1 mM copper sulfate (aqueous) at a flowrate of 1 mL/min, and a 254 nm observation wavelength.
Identification and purity were assessed by NMR and matched the reported literature 1 .
N-(diphenyl)glycine tert-butyl ester (2) was synthesized according to published procedure. Identification and purity were assessed by NMR and matched the reported literature 1 .
Following the addition, the mixture was stirred for 15 min. The reaction mixture was passed through a silica light sep pak and collected as Fraction 1. MeCN (1 mL) was used to rinse the reaction vessel and passed through the silica light sep pak and collected as Fraction 2. TFA (1 mL) was added to Fraction 2 and the mixture was
Following the addition, the mixture was stirred for 15 min. The reaction mixture was passed through a silica light sep pak and collected as  with 0.5 mL of 0.9% NaCl. The Accel Plus CM cartridge was subsequently removed from the module to be eluted in 0.5 mL incremental fractions with 1 mL of sodium citrate followed by 3 mL of 0.9% NaCl.