3-Azido-D-alanine

Alanizi, Aryn A., et al. Bioconjugate Chemistry 35.4 (2024): 517-527.

Imaging bacterial infections with PET requires tracers that selectively mark bacteria over host tissue. Incorporation of d-amino acids into bacterial peptidoglycan provides a metabolic route to introduce bioorthogonal handles; 3-azido-D-alanine is an azide-bearing d-amino acid designed to become covalently installed into bacterial cell-wall muropeptides and serve as a reactive handle for strain-promoted alkyne-azide cycloaddition (SPAAC).
Application and Methodology: In this methodology, live pathogenic bacteria, including Staphylococcus aureus, were first incubated with 3-Azido-D-alanine. This allowed the azide-functionalized amino acid to be metabolically integrated into the bacterial cell wall. Subsequently, a water-soluble, [18F]-labeled radiotracer containing a dibenzocyclooctyne (DBCO) group ([18F]FB-sulfo-DBCO) was introduced. The DBCO group undergoes a rapid and selective strain-promoted azide-alkyne cycloaddition (SPAAC) "click" reaction with the azide groups presented on the bacteria, thereby covalently attaching the radionuclide.
Key Findings:
· [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity and functioned as an effective SPAAC reporter for azide-labeled bacterial cell walls.
· Bacteria treated with 3-azido-D-alanine showed significantly higher radiotracer ligation than control cultures treated with D-alanine; for example, Staphylococcus aureus cultures exhibited ~7-fold greater [18F]FB-sulfo-DBCO incorporation when pretreated with 3-azido-D-alanine.
· The tracer's in vivo biodistribution in healthy mice was assessed to provide baseline pharmacokinetic information relevant to eventual infection imaging studies.

Soto, Paul, et al. Materials Today Bio. (2022): 19.

Magnetospirillum gryphiswaldense (MSR-1) is a model magnetotactic bacterium (MTB) that biomineralizes magnetosome chains and is used in biohybrid and living-material applications. Introducing an azide into bacterial biomolecules via metabolic labeling enables bioorthogonal attachment of cargos by SPAAC or other click reactions; 3-Azido-D-alanine is a D-amino-acid analogue intended to incorporate into peptidoglycan and present cell-surface azides for downstream conjugation.
Approach: MSR-1 cultures were started at low density (OD600 ≈ 0.02) and grown for three days at 28 °C under microoxic conditions (2% O2) in Hungate tubes using modified Flask Standard Medium with 50 µM ferric iron citrate. The modified amino acid was added immediately after inoculation. Three concentrations of 3-Azido-D-alanine were tested: 0.005 mM, 0.060 mM, and 0.100 mM.
Key Results:
· Concentration-dependent effects: TEM suggested fewer cells with intact magnetosome chains as the concentration of 3-Azido-D-alanine increased. Cmag measurements showed a decline in the population magnetic signal with higher amino-acid dosing.
· Physiological stress at higher doses: Optical microscopy revealed shape changes and larger, non-dividing cells at the highest concentration (0.100 mM), indicative of stress; nevertheless, cells across conditions remained motile.
· Growth parameters: Across tested batches, generation/doubling times calculated from growth curves ranged roughly 4.5-6.75 hours.
· The data indicate a practical upper limit for 3-Azido-D-alanine supplementation above which magnetosome biogenesis and normal cell physiology are compromised.