L-Azidohomoalanine hydrochloride

Shalizi, Aryaman, et al. Plos one 15.6 (2020): e0234542.

Bloodstream infections caused by Staphylococcus aureus, which can progress to life-threatening sepsis, require rapid diagnosis for effective treatment. This study developed a "Click-to-Capture" workflow based on bio-orthogonal chemistry. The core of this strategy was the use of L-Azidohomoalanine (LAHA), a synthetic amino acid analog bearing an azide group. This compound is metabolically incorporated by bacteria into newly synthesized cell surface proteins, thereby displaying bio-orthogonal azide handles on the outer surface of the bacterial cells.
Methodology: In this method, a sample containing S. aureus is incubated with LAHA for a short duration. The bacteria metabolically incorporate LAHA into their surface proteins. Subsequently, a cyclooctyne-biotin reagent is added, which rapidly and selectively "clicks" with the azide groups presented on the bacterial surface via a copper-free cycloaddition reaction. The biotinylated bacteria are then efficiently captured using streptavidin-coated beads, enabling their purification from the sample matrix.
Key Results: The use of LAHA proved highly effective for rapid labeling. The entire enrichment workflow, from the crude sample to captured bacteria, was completed within one hour. Bacteria enriched using this LAHA-based method remained viable and were suitable for subsequent culture or molecular assays. The study demonstrated successful capture of S. aureus across a clinically relevant concentration range (50-10^4 CFU/mL). Furthermore, the method was shown to be significantly faster than existing bio-orthogonal approaches that require hours of labeling time and is applicable to Gram-positive organisms like S. aureus.

Wang, Jigang, et al. Nature protocols 12.2 (2017): 279-288.

Autophagy is a primary catabolic route responsible for the degradation of long-lived proteins and organelles. Robust measurement of autophagic flux is necessary to further characterize cell physiology and disease. L-Azidohomoalanine (AHA) is a surrogate of methionine that is metabolically incorporated into actively translating proteins and contains an azide "click" handle that can be conjugated to a fluorescent alkyne in the presence of a copper catalyst. This chemical reaction is the basis for a nonradioactive pulse-chase method that can be used to quantify the degradation of long-lived proteins as a measure of autophagic flux.
Protocol Design Using AHA:
· Pulse Labeling: Cells are cultured in methionine-free medium supplemented with AHA for 18 hours, where AHA is metabolically incorporated into all newly synthesized proteins.
· Chase Phase: Chase short-lived proteins by culturing cells in complete DMEM supplemented with L-methionine; this also minimizes reincorporation of released AHA during subsequent starvation.
· Induction and Detection: Fix and permeabilize cells, then perform the click reaction between the AHA azide and a fluorescent alkyne probe (e.g., TAMRA-alkyne, TCEP, TBTA ligand, CuSO4). Analyze fluorescence by flow cytometry.
Key Results: The AHA-based method successfully provided a quantitative measure of autophagic degradation. In mouse embryonic fibroblasts (MEFs), cells subjected to autophagy-inducing starvation showed a measurable decrease in fluorescence intensity compared to control cells. A typical result was a 20% reduction in fluorescence after 3 hours of starvation, directly representing the level of long-lived protein degradation via autophagy.