Cy3 methyltetrazine (TZ-Cy3) is a reagent for click chemistry with a methyltetrazine structural unit that is highly reactive toward cyclooctene. Cy3 methyltetrazine is also a tetrazine-modified fluorescent probe that may be utilized to analyze protein phosphorylation in solution and living cells.

Physicochemical Properties

Molecular Formula	C40H46BF4N7O
Molecular Weight	727.64
Exact Mass	727.379
CAS #	2183473-57-4
PubChem CID	154704808
Appearance	Typically exists as solid at room temperature
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	11
Rotatable Bond Count	11
Heavy Atom Count	53
Complexity	1210
Defined Atom Stereocenter Count	0
SMILES	[B-](F)(F)(F)F.CC1=NN=C(N=N1)C2=CC=C(C=C2)CNC(=O)CCCCCN\3C4=CC=CC=C4C(/C3=C/C=C/C5=[N+](C6=CC=CC=C6C5(C)C)C)(C)C
InChi Key	XMPUYESTAOFGCZ-UHFFFAOYSA-O
InChi Code	InChI=1S/C40H45N7O.BF4/c1-28-42-44-38(45-43-28)30-24-22-29(23-25-30)27-41-37(48)21-8-7-13-26-47-34-18-12-10-16-32(34)40(4,5)36(47)20-14-19-35-39(2,3)31-15-9-11-17-33(31)46(35)6;2-1(3,4)5/h9-12,14-20,22-25H,7-8,13,21,26-27H2,1-6H3;/q;-1/p+1
Chemical Name	6-[(2Z)-3,3-dimethyl-2-[(E)-3-(1,3,3-trimethylindol-1-ium-2-yl)prop-2-enylidene]indol-1-yl]-N-[[4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl]methyl]hexanamide;tetrafluoroborate
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro

Guidelines (This is our suggested protocol; it should be adjusted based on your unique requirements as it just offers as a guideline). Confocal microscopy of cellular phosphorylation: 1. Use H1299 cells as an example and culture them for 24 hours in a coverglass-bottom imaging dish. 2. For one hour, incubate H1299 cells in varying doses of ATP-NB (an ATP analogue functionalized by norbornene that may efficiently phosphorylate proteins in living cells through cell penetration). 3. To add fluorescence to the phosphorylated protein, add Cy3 methyltetrazine. 4. For one hour, incubate in a new medium. 5. To liberate the Cy3 methyltetrazine probe, wash the cells twice in PBS. 6. Incubate the cells for 15 minutes at room temperature with 4% paraformaldehyde and for 10 minutes at room temperature with 0.1% Triton X-100 solution. Use PBS to wash cells four times. 8. To produce the high-resolution images of cells (Ex=488 nm; Em=561 nm), use a confocal microscopy imaging device.

References

[1]. Analysis of protein phosphorylation in solution and in cells by using an ATP analogue in combination with fluorescence techniques. Analyst. 2021 Jul 21;146(14):4506-4514.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.3743 mL	6.8715 mL	13.7431 mL
	5 mM	0.2749 mL	1.3743 mL	2.7486 mL
	10 mM	0.1374 mL	0.6872 mL	1.3743 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.