1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane: Use & Handling Guide

1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane is a short-chain PEG-based azide reagent widely used as a hydrophilic spacer and reactive handle in copper-catalyzed and strain-promoted azide–alkyne cycloaddition (click chemistry) bioconjugation workflows.

CAS Number

167961-08-2

Molecular Formula

C7H15N3O3

Molecular Weight

189.21 g/mol

Aladdin SKU

A487268

Reactive Group

Terminal azide (–N₃)

Solubility

Miscible with water, DMF, DMSO, DCM, and most common organic solvents

Storage Condition

Store at 2–8 °C, protected from light and heat, under inert atmosphere

Chemical Identity and Properties

1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane (SKU A487268; CAS 167961-08-2) is a low-molecular-weight, triethylene glycol-derived azide building block with the molecular formula C₇H₁₅N₃O₃ and a molecular weight of 189.21 g/mol. The compound consists of a terminal azide group (–N₃) tethered to a three-unit ethylene oxide chain capped with a methyl ether at the distal end.

The PEG backbone confers significant aqueous solubility and reduces non-specific hydrophobic interactions, making this reagent particularly valuable when hydrophilicity must be preserved in the final conjugate. The terminal azide is the sole reactive functional group under ambient conditions, remaining inert to most nucleophiles, electrophiles, and biological functional groups until activated by an appropriate dipolarophile (alkyne or cyclooctyne).

Key physicochemical characteristics:

• Appearance: colorless to pale yellow liquid
• Molecular weight: 189.21 g/mol
• Solubility: miscible with water, DMSO, DMF, DCM, THF, and alcohols
• Boiling point and density: consult the product SDS for confirmed values

Applications in Click Chemistry and Bioconjugation

The terminal azide of 1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane participates in two major bioorthogonal ligation strategies:

1. Copper-Catalyzed Azide–Alkyne Cycloaddition (CuAAC)

In the presence of Cu(I) catalyst (commonly generated in situ from CuSO₄ and sodium ascorbate), the azide reacts selectively and quantitatively with terminal alkynes to form a 1,4-disubstituted 1,2,3-triazole linkage. This reaction proceeds at room temperature in aqueous or mixed aqueous–organic media and is highly tolerant of diverse functional groups present in biological molecules.

2. Strain-Promoted Azide–Alkyne Cycloaddition (SPAAC)

For copper-free, metal-free bioconjugation—essential when working with live cells, sensitive proteins, or in vivo systems—the azide reacts efficiently with strained cyclooctynes (e.g., DIBO, DBCO, BCN). This copper-free variant avoids cytotoxicity associated with Cu(I) and is fully bioorthogonal.

Representative Uses

• Surface functionalization: introduction of hydrophilic azide handles onto nanoparticles, gold surfaces, or polymer scaffolds for subsequent click ligation
• PEGylation mimic: the short PEG chain improves solubility and reduces immunogenicity of small-molecule drug conjugates
• Labeling and imaging: attachment of azide-bearing PEG spacers to biomolecules prior to fluorophore or affinity-tag ligation
• Linker in ADCs and PROTACs: serves as a hydrophilic tether component in complex multi-step synthesis

Safe Handling and Storage Protocol

Low-molecular-weight aliphatic azides are classified as potentially shock- and heat-sensitive compounds. Although 1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane is less hazardous than simple alkyl azides due to the stabilizing influence of the oxygen-rich PEG chain, standard precautions for organic azides must be observed at all times.

Personal Protective Equipment (PPE)

• Wear chemical-splash goggles, a laboratory coat, and nitrile gloves at minimum
• Work in a well-ventilated fume hood; avoid open-bench handling
• Use a face shield when handling larger quantities or concentrated solutions

Handling Precautions

• Keep away from open flames, sparks, and all ignition sources; the compound is flammable
• Avoid exposure to strong acids, reducing agents, and transition-metal catalysts in the absence of controlled reaction conditions—these can trigger decomposition or uncontrolled reduction of the azide
• Do not allow the neat liquid or concentrated solutions to contact metal surfaces that may catalyze decomposition
• Handle in small quantities; do not distill or heat the neat compound to elevated temperatures

Storage

• Store at 2–8 °C in a tightly sealed container under an inert atmosphere (nitrogen or argon)
• Protect from light, moisture, and heat sources
• Keep away from incompatible materials: strong oxidizers, strong reducing agents, and heavy metal salts
• Check container integrity before each use; discard if discoloration or precipitate is observed

Waste Disposal

Dispose of azide-containing waste in accordance with local, institutional, and national regulations. Do not pour neat azide reagents into metal drains; quench with aqueous sodium nitrite/acetic acid or consult your institution's chemical waste management program. Always refer to the current Safety Data Sheet (SDS) before use.

Recommended Reaction Protocols

The following protocols outline general best-practice procedures. Specific conditions should be optimized for each substrate and application.

Protocol A: CuAAC Ligation in Aqueous Buffer

1. Prepare a stock solution of the azide reagent (A487268) in DMSO or water at 10–100 mM. Vortex briefly to ensure complete dissolution.
2. Prepare alkyne-bearing substrate in the reaction buffer (e.g., PBS pH 7.4 or HEPES pH 7.0–7.5).
3. In the reaction vessel combine: alkyne substrate, azide reagent (1.2–5 equivalents relative to alkyne), CuSO₄ (0.1–1 equiv), sodium ascorbate (5–10 equiv relative to Cu), and optionally a Cu-stabilizing ligand such as THPTA or BTTAA (2–5 equiv relative to Cu) to protect sensitive biomolecules.
4. Adjust volume with buffer; mix gently under inert atmosphere.
5. React at room temperature for 30 min to 4 h with gentle agitation, monitoring by LC-MS or TLC.
6. Quench by adding EDTA (10 equiv relative to Cu) to chelate residual copper; purify product by HPLC, size-exclusion chromatography, or dialysis as appropriate.

Protocol B: SPAAC Ligation (Copper-Free)

1. Prepare azide reagent stock as above.
2. Combine azide reagent (1.2–3 equivalents) with cyclooctyne-functionalized substrate (e.g., DBCO- or BCN-conjugate) in PBS or cell culture medium.
3. React at room temperature or 37 °C for 1–16 h depending on the cyclooctyne reactivity and required conversion.
4. No quench step is required; proceed directly to purification or analysis.

General Tips

• Use degassed solvents to minimize oxidation of Cu(I) in CuAAC reactions.
• Perform reactions in amber vials or wrapped in foil to protect azide from prolonged UV exposure.
• Confirm complete reaction by LC-MS: the triazole product will show characteristic mass shift relative to starting materials.

Quality, Purity, and Analytical Verification

Aladdin Scientific supplies 1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane (A487268) with a certificate of analysis (CoA) specifying purity determined by appropriate analytical methods. Researchers are encouraged to verify purity upon receipt and before use in critical experiments.

Recommended Analytical Methods

• ¹H NMR and ¹³C NMR: Confirm structural identity. Key signals include the azide-adjacent –CH₂– methylene protons (typically δ 3.2–3.5 ppm) and the terminal methoxy singlet (δ ~3.3 ppm).
• IR Spectroscopy: The strong asymmetric azide stretch at ~2095–2100 cm⁻¹ is diagnostic for the intact –N₃ group.
• GC-MS or LC-MS: Confirm molecular ion at m/z 189.21 (M) or corresponding adducts; assess purity by peak area integration.
• Refractive index or density: Physical constants can be cross-referenced with literature or SDS values to confirm identity.

Purity Acceptance Criteria

For most synthetic applications, ≥95% purity by GC or HPLC area normalization is acceptable. For sensitive biological assays or in vivo use, ≥98% purity is recommended. Consult the CoA supplied with each lot for confirmed purity values. Contact Aladdin Scientific technical support for custom purity requirements.

Stability Monitoring

Azide compounds can degrade upon prolonged storage, exposure to light, or temperature cycling. Monitor for any yellowing, turbidity, or change in IR azide stretch intensity as indicators of degradation. If degradation is suspected, do not use the reagent in critical experiments without re-analysis.

Frequently asked questions

What is the difference between using this azide in CuAAC versus SPAAC reactions?

CuAAC requires a Cu(I) catalyst and a terminal alkyne partner, providing fast and high-yielding ligation but introducing copper that may be cytotoxic and must be removed from biological samples. SPAAC uses strained cyclooctynes (e.g., DBCO, BCN) and requires no metal catalyst, making it compatible with live cells and in vivo applications, though reaction rates are generally slower and the cyclooctyne reagents are larger and more costly.

How should I prepare a working stock solution of 1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane?

Allow the vial to equilibrate to room temperature before opening to prevent condensation. Dissolve the reagent in anhydrous DMSO or water to prepare a 10–100 mM stock solution. Vortex briefly to ensure homogeneity, aliquot into single-use portions to avoid repeated freeze–thaw cycles, and store aliquots at –20 °C under inert gas if long-term storage is needed.

Is 1-Azido-2-(2-(2-methoxyethoxy)ethoxy)ethane compatible with biological buffers and cell culture media?

Yes, the compound is water-miscible and the hydrophilic PEG backbone ensures good aqueous compatibility. It is stable in common biological buffers such as PBS and HEPES at physiological pH and can be diluted directly into cell culture media for SPAAC-based labeling experiments. For CuAAC in biological systems, use Cu-chelating ligands to minimize free Cu(I) toxicity.

What safety precautions are most critical when working with this organic azide?

Always work in a fume hood wearing appropriate PPE (goggles, gloves, lab coat). Keep the reagent away from heat, ignition sources, strong acids, and heavy metal salts that could trigger decomposition. Never distill or heat the neat compound. Dispose of azide waste according to institutional regulations, using appropriate chemical quench procedures rather than pouring down metal drains.

How can I confirm the azide group is intact in my stock solution before use?

The most rapid and reliable method is ATR-FTIR or transmission IR spectroscopy: an intact azide will show a strong, sharp absorption band near 2095–2100 cm⁻¹. Absence or significant reduction of this band indicates degradation. LC-MS can also confirm the molecular weight and purity of the compound prior to use.

References

1. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition. 2001, 40, 2004–2021. doi:10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5
2. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. A Strain-Promoted [3 + 2] Azide–Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. Journal of the American Chemical Society. 2004, 126, 15046–15047. doi:10.1021/ja044996f
3. Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Analysis and Optimization of Copper-Catalyzed Azide–Alkyne Cycloaddition for Bioconjugation. Angewandte Chemie International Edition. 2009, 48, 9879–9883. doi:10.1002/anie.200905087

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.