1-Azidoethylcholine Bromide

Choline phospholipids labeling agent, visualizing the subcellular localization of choline phospholipids.

What is it?

• It is a choline analogue: the molecule mimics the head-group of choline (which is incorporated into phospholipids like phosphatidylcholine). ResearchGate+3salic.med.harvard.edu

• It has an azido (-N₃) group attached (hence “azidoethyl-choline”) which renders it “clickable” (able to participate in bioorthogonal chemistry). salic.med.harvard.edu+2

• One salt variant is the bromide form. aobious.com+1

What is the functionality / how is it used?

Here are the main functionalities and applications:

1. Metabolic incorporation into choline-containing phospholipids

   • Cells naturally take up choline, activate it (via phosphorylation, CDP-choline path) and incorporate it into phosphatidylcholine (PC) and related lipids. PMC+1

   • The azidocholine analogue is taken up similarly, incorporated into choline-phospholipids (for example into PC) such that the resulting lipid bears the azide tag. For example: AECho → “azidoethyl-phosphatidylcholine” in cells. salic.med.harvard.edu+1

   • Because the analogue is tolerated (i.e., doesn’t majorly perturb normal lipid pools) and integrates into the membranes, it gives a way to trace or image choline-lipid metabolism/dynamics. salic.med.harvard.edu+1

2. Bioorthogonal “click chemistry” labelling

   • Once the azide-tagged lipid is in place in a cell or organism, one can apply a complementary reagent (for example alkyne-fluorophore, dibenzocyclooctyne (DBCO)-fluorophore, etc) to “click” onto the azide and thus tag the lipid with a fluorophore or probe. salic.med.harvard.edu+1

   • This enables visualization of the labeled lipids: imaging their location (cell membrane surface, organelles), their trafficking, dynamics. For example the 2015 study by Jao et al. showed localization to mitochondria, internal membranes etc. salic.med.harvard.edu

   • It also opens possibility for two-colour labelling: e.g., combining azidocholine (azide) with propargyl-choline (alkyne) and then using orthogonal click reactions to differentiate two pools. salic.med.harvard.edu+1

3. Monitoring phospholipid synthesis / cellular membrane studies

   • Because it labels choline-phospholipids (which are abundant and functionally critical in membranes), this reagent is used in studies of membrane biology, lipid metabolism, membrane trafficking, organelle biology. For example: monitoring choline-containing phospholipid synthesis in cell culture and even in whole organisms. 

   • More recently, it has been used in bacterial studies: e.g., metabolic labeling in bacteria (gram negative) to assess outer-membrane integrity via phospholipid externalization. ScienceDirect+1

Key properties

• Molecular formula (for the cation): C₆H₁₅N₄O (for the iodo salt, but similar for bromide)

• Molecular weight: ~159.21 g/mol for the cation in some form. 

• Solubility: Water / PBS buffer. 

• Storage: Typically –20 °C, avoid freeze/thaw. 

• Purity: ≥ 95% (H NMR confirms) for reagent quality.

Advantages & strengths

• Allows specific labelling of choline-headgroup lipids with minimal perturbation of the natural lipidome (in the right conditions) — the Jao et al. paper found that AECho was incorporated without grossly changing non-choline phospholipids. salic.med.harvard.edu

• Enables high‐resolution imaging by combining metabolic labeling + click chemistry + fluorescence. Gives insight into localization, trafficking of choline lipids in live or fixed cells.

• Because it uses the endogenous metabolism of choline, it can serve as a reporter of biosynthetic activity of choline lipids, or changes in that metabolism.

• The azide functionality is bioorthogonal (i.e., minimally interfering with native biology) which means the system can be relatively benign.

Limitations / considerations

• Since it is a modified analogue of choline, if used at too high concentrations it could perturb the lipid metabolism or cell physiology. One must optimize labelling concentration and time. (Indeed the literature assessed perturbation of lipid pools). salic.med.harvard.edu

• The click reaction (e.g., DBCO or SPAAC) requires reagents and perhaps fixation or specific conditions; live‐cell compatibility may have constraints (e.g., cytotoxicity of reagents, background labelling).

• Because it competes with endogenous choline, if choline levels are high or the analogue uptake is low, the incorporation may be less efficient. For example, Jao et al. noted ~20% replacement of total choline‐PC at high analogue conc and 24 h. salic.med.harvard.edu

• It labels choline-containing phospholipids, so it’s specific to that class; it does not label all lipids. One must interpret accordingly.

• It is a research reagent (RUO – research use only) and not intended for therapeutic or clinical diagnostic use.

Example use‐case

From Jao et al. (2015): They incubated mammalian cells with AECho, then isolated lipids, and showed by mass spectrometry that the analogue was incorporated into phosphatidylcholine classes without disturbing other phospholipids. They then used a fluorescent DBCO–Alexa488 reagent to click onto the azide and imaged sub‐cellular localisation: cell surface membranes, organelles, mitochondria. salic.med.harvard.edu
Another study in bacteria used AECho metabolic labelling as a probe of outer‐membrane (OM) integrity: in mutants with compromised OM, the labelled phospholipids became accessible to the fluorescent click reagent, enabling detection of membrane defects. ScienceDirect

Practical tips for lab use

• Optimize concentration and incubation time: too low → weak labelling; too high → possible perturbation.

• Ensure the click reagent is compatible (e.g., strain-promoted alkyne reagent if doing live‐cell, to avoid copper toxicity).

• Consider controls: unlabeled cells, choline competition, or blocking uptake to assess specificity.

• After labelling, one can analyse by fluorescence microscopy, flow cytometry, or lipidomic (mass spec) analysis.

• For imaging, ensure the fluorophore choice and imaging conditions match the expected membrane localisation (e.g., exclude nuclear signal, verify membrane vs cytosol).

• Because the reagent is “head‐group” analogue of choline, confirm that downstream functional perturbation is minimal in your cell system (especially if studying metabolic/structural lipid biology).