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| Targets |
Alkyne-tagged proteins, peptides, nucleic acids, and other biomolecules via click chemistry. Cyanine3 azide chloride does not have a direct biological target but rather serves as a chemical probe that covalently binds to molecules containing alkyne groups through CuAAc or SPAAC reactions. The Cy3 fluorophore provides strong fluorescent signal for detection and imaging, while the azide group provides specificity for click chemistry labeling. This reagent is used to track and visualize alkyne-labeled targets in various biological assays, including protein localization, protein-protein interactions, and nucleic acid detection.
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| ln Vitro |
In vitro, Cyanine3 azide chloride is used to label alkyne-functionalized biomolecules. At concentrations of 1-50 uM, it efficiently conjugates to alkyne-modified proteins via CuAAc (copper sulfate, tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), and sodium ascorbate) within 1-2 hours at 37degC. The labeled proteins retain their biological activity and can be detected by fluorescence spectroscopy, SDS-PAGE with in-gel fluorescence scanning, Western blotting, or immunofluorescence. The dye is also used in metabolic labeling experiments where cells are cultured with alkyne-modified sugars (e.g., GlcNAz) or amino acids (e.g., AHA), followed by click chemistry labeling of newly synthesized glycoproteins or nascent proteins. Flow cytometry and fluorescence microscopy are used to detect and quantify labeled cells.
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| ln Vivo |
In vivo applications of Cyanine3 azide chloride are limited due to the need for copper catalysts for CuAAc reactions, which are toxic to living organisms. However, the dye can be used in ex vivo labeling of tissues or cells following bioorthogonal metabolic labeling. In animals administered with alkyne-modified precursors (e.g., azido sugars), harvested tissues can be labeled with Cyanine3 azide chloride ex vivo to visualize the distribution of labeled biomolecules. The strain-promoted SPAAC reaction (which does not require copper) offers potential for in vivo click chemistry applications, where DBCO- or BCN-functionalized targeting molecules can be conjugated to Cy3 azide in living animals. Detailed in vivo activity data is limited as the compound is primarily used in vitro or ex vivo.
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| Enzyme Assay |
In a cell-free click chemistry reaction, Cyanine3 azide chloride is mixed with an alkyne-containing substrate (e.g., alkyne-modified BSA, alkyne-containing small molecule) in PBS or other suitable buffer. For CuAAc, a reaction mixture containing 10-100 uM Cyanine3 azide, 10-100 uM alkyne substrate, 100 uM CuSO4, 500 uM THPTA, and 5 mM sodium ascorbate is incubated at room temperature or 37degC for 1-2 hours. The reaction is stopped by the addition of EDTA (5-10 mM). The labeled product is then purified by size exclusion chromatography, dialysis, or precipitation to remove unreacted dye. Conjugation efficiency is assessed by SDS-PAGE followed by fluorescence scanning (excitation at 550-580 nm, emission at 570-620 nm) or by mass spectrometry. For SPAAC, the reaction proceeds without copper by mixing Cyanine3 azide (10-50 uM) with a DBCO- or BCN-modified substrate in PBS at 37degC for 2-24 hours.
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| Cell Assay |
For cell labeling experiments, cells are first cultured with an alkyne-modified precursor (e.g., 25-100 uM alkyne-sugar or alkyne-amino acid) for 24-72 hours to incorporate the alkyne tag into cellular biomolecules. After labeling, cells are washed with PBS, fixed with 4% paraformaldehyde for 15-20 minutes, and permeabilized with 0.1-0.5% Triton X-100 if necessary. The click chemistry reaction mixture (Cyanine3 azide chloride 10-50 uM, CuSO4 100-200 uM, THPTA 200-500 uM, sodium ascorbate 2-5 mM) is prepared fresh and added to cells. The reaction is carried out at room temperature or 37degC for 1-2 hours, protected from light. After click labeling, cells are washed 3 times with PBS to remove unreacted dye. Labeled cells are then imaged by fluorescence microscopy (excitation at 550-570 nm, emission at 570-610 nm) or analyzed by flow cytometry using a 561 nm or 640 nm laser and appropriate emission filters (PE-Cy5 or APC channel).
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| Animal Protocol |
For in vivo experiments that utilize SPAAC (no copper), mice bearing tumors or with specific targeting constructs receive an intravenous injection of DBCO- or BCN-modified targeting molecule (1-5 mg/kg). After allowing 4-24 hours for target binding and circulation, mice receive a second intravenous injection of Cyanine3 azide chloride (0.5-2 mg/kg in PBS or 5% DMSO/PBS). Click conjugation occurs in vivo through SPAAC without the need for toxic catalysts. Mice are anesthetized with isoflurane and imaged 1-24 hours later using an in vivo imaging system with excitation at 640 nm and emission at 700-720 nm. Tissues are harvested for ex vivo fluorescence imaging and histological analysis. Alternatively, for copper-dependent labeling, animals are sacrificed and tissues are fixed, then subjected to ex vivo click chemistry labeling as described in the cellular protocol.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for Cyanine3 azide chloride is limited due to its primary use as a labeling reagent rather than a therapeutic agent. After intravenous administration, the compound is expected to have a short circulation half-life of 5-15 minutes due to rapid clearance by the kidneys and metabolism. The compound is moderately soluble in organic solvents (DMF, DMSO, dichloromethane) but practically insoluble in water (~0.04 mg/mL). The logP value is estimated to be between 2-4, indicating moderate lipophilicity. The azide group is chemically stable in aqueous solutions but may be reduced in the presence of reducing agents (e.g., dithiothreitol, beta-mercaptoethanol). The Cy3 fluorophore is resistant to photobleaching and exhibits good quantum yield (~0.31). For detailed quantitative PK data, users are advised to consult custom pharmacokinetic studies or perform their own assessments.
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| Toxicity/Toxicokinetics |
Toxicological data for Cyanine3 azide chloride indicates that it is for research use only and not intended for human use. The compound is an irritant and may cause skin, eye, and respiratory tract irritation upon contact or inhalation. Acute toxicity studies are limited, but the compound should be handled with caution. The copper required for CuAAc reactions is toxic to cells and animals, and care should be taken to remove copper by extensive washing when used in vitro. The azide group is potentially explosive when heated or subjected to shock, although at the milligram scale used in research, the risk is minimal. Standard precautions for handling reactive chemicals and fluorophores should be observed, including the use of gloves, eye protection, and a fume hood. The compound is not listed as a hazardous substance by major regulatory agencies at the small quantities used in research, but local regulations may vary.
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| References | |
| Additional Infomation |
Cyanine3 azide chloride is a research-grade click chemistry reagent with no approved clinical applications. It is supplied as a green to dark green solid powder with a purity of ≥95%. The compound is soluble in organic solvents (DMF, DMSO, dichloromethane) but has limited aqueous solubility (0.04 mg/mL). It should be stored at -20degC, protected from light and moisture, for up to 3 years as a powder. Stock solutions in DMSO can be stored at -20degC for up to 6 months, but avoid repeated freeze-thaw cycles. Cyanine3 azide chloride is a powerful tool for bioorthogonal labeling, enabling specific and stable conjugation to alkyne-modified biomolecules without interfering with biological function. It is widely used in fluorescence microscopy, flow cytometry, and Western blotting for protein detection, as well as in glycobiology and proteomics research. Synonyms include Cy3 azide, Cy3-N3, and N-(3-azidopropyl)-6-[(2E)-3,3-dimethyl...
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| Molecular Formula |
C44H53CLN6O
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| Molecular Weight |
717.3842
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| Exact Mass |
574.318
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| CAS # |
1167421-28-4
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| PubChem CID |
124080898
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| Appearance |
Green to dark green solid powder
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
41
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| Complexity |
1030
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C12C=CC=CC1=C1C(C)(C)/C(=C\C=C\C3C(C)(C)C4C5C=CC=CC=5C=CC=4[N+]=3CCCCCC(=O)NCCCN=[N+]=[N-])/N(CCCC)C1=CC=2.[Cl-]
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| InChi Key |
JWFYJJIXFMMPNH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C33H42N6O.ClH/c1-32(2)25-15-8-10-17-27(25)38(5)29(32)19-13-20-30-33(3,4)26-16-9-11-18-28(26)39(30)24-12-6-7-21-31(40)35-22-14-23-36-37-34;/h8-11,13,15-20H,6-7,12,14,21-24H2,1-5H3;1H
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| Chemical Name |
N-(3-azidopropyl)-6-[(2E)-3,3-dimethyl-2-[(E)-3-(1,3,3-trimethylindol-1-ium-2-yl)prop-2-enylidene]indol-1-yl]hexanamide;chloride
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3940 mL | 6.9698 mL | 13.9396 mL | |
| 5 mM | 0.2788 mL | 1.3940 mL | 2.7879 mL | |
| 10 mM | 0.1394 mL | 0.6970 mL | 1.3940 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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