FAM azide, 6-isomer  (Synonyms: 6-FAM azide)

FAM azide, 6-isomer (6-FAM azide) is a 6-carboxyfluorescein derivative with an azide functional group and coupling partner in copper-catalyzed azide-alkyne cycloaddition. FAM azide, 6-isomer participates in synthesis of fluorescently tagged disparlure enantiomers for pheromone-binding protein binding assays. FAM azide, 6-isomer is widely used for labeling oligonucleotides (Ex/Em = 493/517 nm)^[1].

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
1. Prepare Working Solutions
1.1 FAM azide, 6-isomer stock solution: Prepare a 10 mM stock solution with anhydrous DMSO. Aliquot the solution and store it at -20°C in the dark; avoid repeated freeze-thaw cycles.
1.2 Other essential reagents:
Copper complex: For example, mix a 100 mM aqueous CuSO₄ solution with a 200 mM aqueous THPTA ligand solution at a volume ratio of 1:2, then vortex to mix thoroughly for later use. As a stabilizing ligand for copper ions, THPTA protects biomolecules and accelerates the reaction.
Reducing agent: 100 mM aqueous sodium ascorbate solution, which requires fresh preparation.
Sample to be labeled: A solution of target molecules with terminal alkynes (such as oligonucleotides, peptides, proteins, etc.).

2. Perform Click Chemistry Reaction
2. Mix the following components in a microcentrifuge tube:
Alkynylated sample to be labeled (e.g., concentration >10 mg/mL);
10 mM FAM azide, 6-isomer stock solution (2-10 molar equivalents in excess relative to alkynes);
Add 5-10 equivalents (relative to dye azide) of the pre-mixed CuSO₄/THPTA solution;
Add 10-30 equivalents (relative to dye azide) of freshly prepared 100 mM sodium ascorbate solution to initiate the reaction;
Vortex briefly to mix, then incubate the reaction at room temperature in the dark for 30-60 minutes. For some samples, extended reaction time or temperature optimization may be required.

3. Post-Reaction Purification
After the reaction completes, separate the labeled product from excess free dye using an appropriate method.
For oligonucleotides and peptides: Purification is achievable via ethanol precipitation or HPLC.
For biomacromolecules such as proteins: Purification is achievable via gel filtration chromatography or dialysis.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

458.42

C₂₄H₁₈N₄O₆

1386385-76-7

Solid

Light yellow to yellow

OC(C=C1OC2=CC(O)=CC=C23)=CC=C1C3(O4)C5=CC(C(NCCCN=[N+]=[N-])=O)=CC=C5C4=O

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Pinnelli GR, et al. Design and Synthesis of Fluorophore-Tagged Disparlure Enantiomers to Study Pheromone Enantiomer Discrimination in the Pheromone-Binding Proteins from the Gypsy Moth, Lymantria dispar. J Chem Ecol. 2022;48(3):312-322.  [Content Brief]